Keywords: FRET: Fluorescence Resonance Energy Transfer, FLIM: Fluorescence Lifetime Imaging Microscopy, WB: Western Blotting, HIV-1: Human Immunodeficience Virus type 1, ZF: zinc finger, eGFP: enhanced Green Fluorescent Protein, IP: immunoprecipitation

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein precursor orchestrates viral particle assembly and directs virus budding by recruiting the cellular endosomal sorting complex required for transport (ESCRT) machinery. This is accomplished by two short motifs in the Cterminal p6 region of Gag, namely PTAP and LYPXnL, referred to as the 'late assembly domains' that bind the cellular sorting factors TSG101 and ALIX. The influence of the NC domain of Gag on p6 PTAP/TSG101 interaction and function was analyzed by immunoprecipitation and FLIM-FRET experiments. Investigating the interaction of Gag mutated in NC and p6 with TSG101 showed that both NC zinc fingers are required to strengthen the Gag-TSG101 interaction. In addition we provide evidences that NC-mediated Gag trafficking is responsible for the docking of TSG101 to the plasma membrane. These findings highlight the key role played by the NC region of Gag in the recruitment of the ESCRT machinery and thus in virions budding.

My sincere gratitude goes to my supervisor Dr. Hugues DE ROCQUIGNY for his continuous support during the PhD program. He taught me all basic things to perform my research e.g. from designing an experiment till its analysis to the outcome. His profound practical skills, immense knowledge and critical but valuable remarks led me to do a good research. Several times, I had extensive discussions with him which resulted in innovative ideas. Apart from research, he was always there to help me in my problems.

I also want to thank Dr. Emmanuel BOUTANT for his help in this project, his availability and responses to all my questions.

A special thanks to Dr. Denis DUJARDIN and Dr. Jean-Luc DARLIX for helping me in the project, for his availability, for discussions and scientific advice. A big thank to Dr. Kamal Kant SHARMA who encouraged me for the writing of my thesis, for his help, support and good humor. Warm thank for Dr. Marc MOUSLI for his kind moral support.

INTRODUCTION 1. Human Immunodeficiency Virus

The human immunodeficiency virus (HIV) is a single-stranded, enveloped RNA lentivirus of the retrovirus family (retroviridae) and is the etiological agent of acquired Immunodeficiency Syndrome (AIDS). The family name retroviridae comes from the concept that they replicate by the reverse transcription of their genomic RNA (gRNA) into a linear double-stranded DNA copy and the subsequent covalent integration of this DNA into the host cell genome [START_REF] Flint | Principles of Virology: Moleculare Biology, Pathogensis, and control of Animal Viruses[END_REF]).

Furthermore, the AIDS is characterized by state of low immunity that leads to several illnesses due to a wide range of opportunistic infections and neoplasms [START_REF] Strauss | Viruses and Human Disease[END_REF][START_REF] Flint | Principles of Virology: Moleculare Biology, Pathogensis, and control of Animal Viruses[END_REF]).

Epidemiology and history of HIV infection

The unique pattern related to outbreak of peumocytis carinii pneumonia (PCP) [START_REF] Gottlieb | Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency[END_REF] and Kaposi sarcoma (KS) [START_REF] Friedman-Kien | Disseminated Kaposi's sarcoma syndrome in young homosexual men[END_REF] in 1981, leads to the first clinical AIDS. The Centers for Disease Control and Prevention (CDC) named these patterns of conditions the AIDS as these conditions of a rare opportunistic infection and neoplasm were known to be associated with severely suppressed immune status.

However, the first clue to AIDS etiology came in 1983 after the isolation of the virus from lymph nodes of a patient suffering with cervical lymphadenopathy. Françoise Barré-Sinoussi, Luc Montagnier, and co-workers found core proteins p25 of this virus immunologically unique, being only immune-precipitated with patient serum, hence they gave the isolate a different name: lymphadenopathy-associated virus (LAV) [START_REF] Barre-Sinoussi | Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)[END_REF]. Followed by this, another similar discovery by research group led by Robert Gallo [START_REF] Gallo | Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS)[END_REF], ICTV recommended the name HIV in 1986 [START_REF] Flint | Principles of Virology: Moleculare Biology, Pathogensis, and control of Animal Viruses[END_REF]).

Genetic variability, grouping and distribution

High mutation and recombination of Reverse transcriptase enzyme is main case of HIV-1 evolution and genetic variability which leads to diverse populations of viral quasi-species in each infected individual, differing by up to 10% [START_REF] Thomson | Molecular epidemiology of HIV-1 genetic forms and its significance for vaccine development and therapy[END_REF]. On the basis of variability in the envelope (env) gene, HIV-1 can be classified into four groups [START_REF] Buonaguro | Human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications[END_REF]): the "Major" group M, which accounts for more than 90% of HIV-1 infections; the "Outlier" group O that appears to be restricted to west-central Africa; the "New" group N, a group discovered in 1998 in Cameroon; and group P that has been identified in 2009 by J.C. Plantier, who isolated a new strain closely related to gorilla simian immunodeficiency virus from a Cameroonian woman [START_REF] Plantier | A new human immunodeficiency virus derived from gorillas[END_REF]. Within group M, there are nine genetically distinct subtypes or clades designated A, B, C, D, F, G, H, J and K, with no E or I groups, which have never been found in a pure form but combined with other subtypes in a recombinant form as A/E or A/I, …etc. HIV-1 subtypes are typically associated with certain geographical regions with the most widespread being subtypes C and A (Figure 1) and the Isolates may differ by 30-40% in the amino acid sequence of the gp120 SU protein [START_REF] Lynch | Appreciating HIV type 1 diversity: subtype differences in Env[END_REF].

Figure 1: Phylogeny of primate lentiviruses. Five discrete groups of primate lentiviruses have so far been identified based on sequence analysis in pol gene. Members within each group share the same genomic organization and are more closely related to one another than to members of different groups. The human isolates are shown in color. For SIVmnd, SIVsyk, and SIVcpz, only one or a few isolates have been characterized and the prevalence in wild populations has not been determined. For HIV-1, HIV-2, SIVagm, and SIVmac, isolates appear to cluster within distinct clades (Nitahara-Kasahara, [START_REF] Nitahara-Kasahara | Novel nuclear import of Vpr promoted by importin alpha is crucial for human immunodeficiency virus type 1 replication in macrophages[END_REF]).

Global trend of HIV infection: an epidemic update

At the end of 2012, the WHO/UNAIDS reported a prevalence of 35.3 million [32.2 million-38.8 million] people estimated to be living with HIV, of which about 430,000 are children below the age of 15. The overall growth of the global AIDS epidemic appears to have been stabilized.

Together with the significant reduction in mortality, the number of people living with HIV worldwide has increased (Figure 2) (WHO 2014). A comprehensive strategy implemented by the World Health organization (WHO) consisting in sustained and effective AIDS health education and health promotion programs, provides further evidence of decreasing incidence and safer sexual behavior among young adults. Accordingly, seven countries showed a statistically significant decline of 25% or more in HIV prevalence. Moreover, the number of people that are now receiving HIV antiretroviral therapy has been increased by 30% by the end of 2012 to reach 5 million worldwide (WHO 2014).

Modes of transmission

The virus can be present in most biological fluids and secretions of the infected patient that contain CD4+ T-lymphocytes, monocytes and macrophages [START_REF] Flint | Principles of Virology: Moleculare Biology, Pathogensis, and control of Animal Viruses[END_REF]. The majority of HIV-1 infections, accounting for about 80%, are acquired through unprotected sexual contacts, either heterosexual (70%) or homosexual (10% of the total percentage). Additionally, the susceptibility of HIV infection increases considerably with the presence of ulcerative genital lesions due to other sexually transmitted diseases [START_REF] Boily | Heterosexual risk of HIV-1 infection per sexual act: systematic review and meta-analysis of observational studies[END_REF]. Second mode of infection is associated with transfusion of contaminated blood and blood products that account for 5% of all transmissions (David L. Heymann. l.. Washington DC 18th (edn) 2004). Lastly, maternal transmission of HIV is also responsible for more than 90% of all HIV infections in infants and children [START_REF] Cavarelli | Human immunodeficiency virus type 1 mother-to-child transmission and prevention: successes and controversies[END_REF].

AIDS, the final stage of HIV infection

Lowering of CD4+ T lymphocyte count to <500 per μl (normal value 600-1200 cells/μl) referred as AIDS related complex (ARC) formation and the loss of cell-mediated immunity. Further decrease in CD4+ T-cell counts (<200 cell/μl) leads the onset of frank AIDS. At this point, the infected individual becomes susceptible to a variety of opportunistic infections and neoplasms and death usually proceeds within an average time interval of 9 months (Figure 3) [START_REF] Pantaleo | New concepts in the immunopathogenesis of human immunodeficiency virus infection[END_REF][START_REF] Fauci | Human immunodeficiency virus (HIV) disease: AIDS and related disorders[END_REF]. During the period following primary infection, HIV disseminates widely in the blood and lymphoid tissue; an abrupt decrease in CD4+ T cells in the peripheral circulation is often seen. An immune response to HIV ensures, with a decrease in detectable viremia. A period of clinical latency follows, during which CD4+ T cells counts continue to decrease, until they fall to a critical level below which there is a substantial risk of opportunistic infections [START_REF] Pantaleo | New concepts in the immunopathogenesis of human immunodeficiency virus infection[END_REF]).

Prevention and control of HIV-1 infection

The CDC suggests post exposure prophylaxis (PEP) for HIV negative person who has recently been exposed to HIV for any reason. The risk of sero-conversion can be reduced by the use of Anti-RetroViral (ARV) drugs for at least four weeks [START_REF] Smith | Antiretroviral postexposure prophylaxis after sexual, injection-drug use, or other nonoccupational exposure to HIV in the United States: recommendations from the U.S. Department of Health and Human Services[END_REF]. Apart from this, the highly active antiretroviral therapy regimen (HAART) reduce considerably HIV morbidity and mortality [START_REF] Adamson | Recent progress in antiretrovirals--lessons from resistance[END_REF]. HAART regimens combine three drugs belonging to at least two classes of the 35 commercially available ARV agents: typically two nucleoside analogues reverse transcriptase inhibitors (NRTIs), plus either a protease inhibitor or non-nucleoside reverse transcriptase inhibitor (NNRTI) [START_REF] Dybul | Guidelines for using antiretroviral agents among HIV-infected adults and adolescents. Recommendations of the Panel on Clinical Practices for Treatment of HIV[END_REF][START_REF] Services | Panel on Antiretroviral Guidelines for Adults and Adolescents.Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents[END_REF]. However, HIV is known for its drug resistance due to high error rate of reverse transcriptase enzyme that represents a major barrier to the development of effective antiviral therapy [START_REF] Adamson | Recent progress in antiretrovirals--lessons from resistance[END_REF]. On the other hand, most ARV drugs are metabolically toxic, with low therapeutic indexes, and cause many adverse effects leading to poor patient compliance and adherence to treatment [START_REF] Carr | Toxicity of antiretroviral therapy and implications for drug development[END_REF][START_REF] Calmy | A new era of antiretroviral drug toxicity[END_REF]).

Next, considerable efforts are being performed in developing a potent vaccine against HIV. In this regard, Virus-Like Particles (VLPs) produced by Gag polyproteins of HIV and SIV have been shown to be potent stimulators of both cellular and humoral immune responses in animal models and therefore can be potentially excellent vaccine candidates [START_REF] Gallo | Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS)[END_REF][START_REF] Jaffray | Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice[END_REF]). However, a novel indicator for the design of a powerful antibodybased HIV vaccine should correlates with the isolation of multiple HIV broadly neutralizing monoclonal antibodies (MCAb) [START_REF] Walker | Broad neutralization coverage of HIV by multiple highly potent antibodies[END_REF] which can be up to 10-100 fold more potent than the recently described PG9, PG16 and VRC01 [START_REF] Binley | Comprehensive cross-clade neutralization analysis of a panel of antihuman immunodeficiency virus type 1 monoclonal antibodies[END_REF][START_REF] Walker | Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target[END_REF][START_REF] Wu | Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1[END_REF][START_REF] Walker | Broad neutralization coverage of HIV by multiple highly potent antibodies[END_REF]).

Viral Life Cycle

The virus targets CD4+ bearing cells, which include dendritic cells, and macrophages and monocytes of the immune system [START_REF] Roitt | Basic concepts and new aspects of vaccine development[END_REF]. The life cycle of HIV-1 starts with the interaction of gp120 viral proteins with the CD4+ receptor on host cell. Next, membrane fusion is promoted by an additional interaction with a chemokine receptor, including CXCR4 and CCR5 [START_REF] Clapham | Immunodeficiency viruses. Spoilt for choice of co-receptors[END_REF]).

Membrane fusion is followed by the entry of the viral core into the cytoplasm. The two strands of viral RNA are then reverse transcribed into DNA by the viral RT. Once synthesized, the viral DNA is transported to the nucleus as part of a pre-integration complex composed of integrase, matrix, RT and Vpr, as well as the cellular host protein HMG-I. Then viral RNA is covalently integrated into the host cell genome by the catalytic activities of integrase [START_REF] Turner | Structural biology of HIV[END_REF]. which is capped at the 5′-end and polyadenylated at the 3′end by the cellular machinery and transported to the ribosomes. Translation of the unspliced RNA produces the viral Gag and, through an occasional read-through, Gag-Pol proteins, which assemble and bud to form immature particles. Proteolytic cleavage of Gag by the viral protease releases the matrix (MA, green), capsid (CA, pink) and nucleocapsid (NC, blue) proteins, which rearrange to form the mature particle [START_REF] Lu | Structural determinants and mechanism of HIV-1 genome packaging[END_REF]).

After integration, the viral genome is transcribed into messenger RNAs (mRNAs) by the host machinery including RNA polymerase II. This RNA polymerase has to be activated by two molecular switches located in two regions at each end of the proviral DNA called long terminal repeats, LTRs. These LTRs are prepared for transcription by binding of NF-ᴋβ/rel protein, which function to increase the transcriptional activity of the gene [START_REF] Greene | AIDS and the immune system[END_REF].

Initially, short spliced RNA transcripts of about 2000 bases in length are produced which code for the regulatory proteins of the virus, e.g. Tat, Rev, and Nef. The Tat protein binds to the trans activation region (TAR) within the viral RNA leading to a phosphorylation of the RNA polymerase by a human kinase [START_REF] Herrmann | Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor[END_REF]. This phosphorylation augments transcription of the proviral DNA by at least 1000-fold. The Nef protein is believed to function in modifying the host cell to make it more suitable for manufacturing the HIV virion [START_REF] Greene | AIDS and the immune system[END_REF]. The third regulatory protein, Rev, is important for switching early phase infection into late stage infection by promoting transport of unspliced RNA transcripts to the cytoplasm [START_REF] Turner | Structural biology of HIV[END_REF]).

In the late stage, two new classes of RNA transcripts appear: long (unspliced) transcripts of 9,200 bases and medium length (singly spliced) of some 4,500 bases. Once the long and medium length transcripts reach the cytoplasm, the cellular machinery begins constructing the components of the new virus. Gag encodes the core proteins, pol encodes reverse transcriptase (RT), protease, integrase, and env encodes the two envelope proteins. In addition three other proteins are encoded for by the vpr, vif, and vpu genes essential for virus replication (see Figure 5). The viral particles then migrate to the outer surface of the cell and obtain their lipid bilayer when budding from the host cell. Subsequent to budding, the Gag polyproteins (hereinafter in this thesis, Gag polyprotein will be referred as Gag) are cleaved by the viral protease to produce the independent enzymes as well as the matrix, capsid, and nucleocapsid protein leading to the formation of the mature virus.

On the basis of literature and observed by cyro-EM, with the maturation of virion, MA remains associated with the viral membrane, GagNC interact with the dimeric gRNA, and CA forms a conical core shell enclosing the NC-RNA complex. This structural reorganization leads to mature infectious virion which disassembles upon entry into a new cell (Figure 4B) [START_REF] Adamson | Human immunodeficiency virus type 1 assembly, release, and maturation[END_REF][START_REF] Briggs | The molecular architecture of HIV[END_REF]).

Structure of the viral particle

Mature HIV is roughly spherical particles measuring about 80-120 nm in diameter, surrounded by a lipoprotein envelope [START_REF] Brugger | The HIV lipidome: a raft with an unusual composition[END_REF]) and studded with ~10 spikes separated from each other by about 22 nm [START_REF] Zhu | Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions[END_REF]. These spikes are composed of a trimer of envelope proteins gp120 [SU] and gp41 [TM] [START_REF] Checkley | HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation[END_REF]. HIV envelope also contain a number of cellular proteins including proteins of MHC I, MHC II, and the intercellular adhesion molecules ICAM1, LFA1, CD28 and CD86 [START_REF] Ott | Cellular proteins detected in HIV-1[END_REF]. Its outer shell is formed of approximately 2000 copies of MA (p17), whereas ~1500-2000 copies of the capsid protein (CA, p24) that form a conical capsid core situated in the center of the viral particle (Fig. 5). This capsid core encloses two copies of the unspliced viral genome which is coated with approximately 2000 copies of the nucleocapsid protein (NCp7), ~10-12 copies of tRNA 3 Lys [START_REF] Isel | Initiation of HIV Reverse Transcription[END_REF], the viral encoded enzymes: protease (PR), reverse transcriptase (RT) and integrase (IN) and a number of viral and cellular proteins [START_REF] Ott | Cellular proteins detected in HIV-1[END_REF]. Above, the HIV-1 genome encodes 3 main genes: gag, pol, and env, as well as the accessory proteins vif, vpu, vpr, tat, rev, and nef. The RNA transcripts that give rise to these proteins are generated as unspliced (gag and pol), singly spliced (env, vif, vpr, vpu) or multiply spliced (tat, rev, nef). For tat and rev, the 5' and 3' exon splice junction sites are marked by a bold line. Sequences found at both the 5' and 3' ends of the genome contain the long-terminal repeats (LTRs), regions that dictate viral RNA synthesis and integration, while genome packaging into new progeny virions is directed by the packaging signal psi at the 5' terminus of the transcript. The HIV-1 genome in this illustration is based on the HXB2 isolate. Below, a schematic of the structural features of the two virus forms: immature (left) and mature (right). The immature virus is characterized by a thick layer of uncleaved gag polyproteins underlying the virion membrane, whereas proteolytic processing of the gag polyproteins during maturation gives rise to a cone-shaped core that encloses the viral RNA and additional viral proteins. This processing step renders the virus infectious. Adapted from (Ganser- [START_REF] Ganser-Pornillos | The structural biology of HIV assembly[END_REF]).

Structural and genetic organization of HIV-1 genome

The proviral DNA

The proviral cDNA is flanked by two LTRs and are divided into 3 domains (Figure 7): U3 (-454 to 1), the repeat sequence R (+1 to +94) and U5 (+95 to +179). The U3 region is further subdivided into: (i) the core promoter (-78 to 1), which harbors the TATA box (-25 to -50), (ii) the activator domain (-104 to -79) and (iii) the modulator domain (-454 to -105), which contains the direct repeat sequence (a stretch of 70-100 nt) that engaged in stimulating the transcription [START_REF] Gaynor | Cellular transcription factors involved in the regulation of HIV-1 gene expression[END_REF][START_REF] Jones | Control of RNA initiation and elongation at the HIV-1 promoter[END_REF][START_REF] Reicin | Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity[END_REF][START_REF] Coffin | Retroviruses Cold Spring Harbor Laboratory Press[END_REF]. Three different classes of mRNA are being transcribed by active gene expression of this proviral cDNA, including the full-length species that forms the viral genome.

The viral genomic RNA

The HIV-1 genome is packaged as two 9,200 nucleotides long single stranded RNAs of positive polarity. Genome is modified post-transcriptionally in a similar way to eukaryotic mRNAs by the addition of a 3'-end tail of 100 to 200 A residues and a 5'-end cap, albeit with a trimethylguanosine (TMG)-capping (Yedavalli and Jeang 2010) (Figure 7). Furthermore, these RNA molecules are associated non-covalently as a dimeric complex in a region near the 5'-end through the DLS (Dimer Linkage Structure) [START_REF] Bender | High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures[END_REF]. Both copies of genomic RNA are full-length RNA, identical in term of cis acting sequences and are packaged as dimer giving rise to pseudo-homozygotes viruses [START_REF] Temin | Genetics of retroviruses[END_REF].This packaging also provides the opportunity for frequent template switching during reverse transcription, resulting in the generation of recombinant viruses genetically distinct from the two parental viruses and directly responsible for the spreading of resistant viruses (Coffin 1979;[START_REF] Hu | Retroviral recombination and reverse transcription[END_REF][START_REF] Temin | Sex and recombination in retroviruses[END_REF][START_REF] Rhodes | High rates of human immunodeficiency virus type 1 recombination: near-random segregation of markers one kilobase apart in one round of viral replication[END_REF].

A. The Non-coding regions

The 5'-untranslated region (UTR) adopts complex secondary and tertiary structures that are involved in key steps of viral replication cycle (Figure 7). Based on extensive in-vitro and exvivo chemical and enzymatic studies, mutational analysis and phylogenetic comparison [START_REF] Harrison | The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure[END_REF][START_REF] Baudin | Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains[END_REF][START_REF] Hayashi | Elucidation of a conserved RNA stem-loop structure in the packaging signal of human immunodeficiency virus type 1[END_REF][START_REF] Clever | Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation[END_REF][START_REF] Damgaard | Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions[END_REF][START_REF] Clever | RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome[END_REF][START_REF] Paillart | First snapshots of the HIV-1 RNA structure in infected cells and in virions[END_REF][START_REF] Wilkinson | High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states[END_REF][START_REF] Watts | Architecture and secondary structure of an entire HIV-1 RNA genome[END_REF], its secondary structure was predicted (Figure 7).

A.1. The R region

The R region (Repeat) (98 nucleotides in HIV-1) is present as two copies of same polarity on both ends of the genome. At 5 ' end, it corresponds to the start site of transcription +1 and the cap site. This highly structured region can further be divided into two sub-domains with different functions:

(i) A stem loop called TAR (Trans-Activating Response element) comprises of 55 to 59 nucleotides (Figure 7). This sequence is important during the first strand transfer of the reverse transcription. It is also involved in the regulation of transcription of viral DNA via the Tat protein and it has been shown to bind with the protein cyclin T1 [START_REF] Rana | Biochemical and functional interactions between HIV-1 Tat protein and TAR RNA[END_REF].

Mutations affecting the sequence or the structure of TAR at its 5' or 3' end, decreases viral replication (Klaver and Berkhout 1994;Klaver and Berkhout 1994;[START_REF] Das | The 5' and 3' TAR elements of human immunodeficiency virus exert effects at several points in the virus life cycle[END_REF]).

(ii) A stem-loop containing the polyadenylation signal (AAUAAA), which is responsible for the addition in 3' poly (A) tail. This tail is necessary for the maturation of mRNA. It has been shown

that stabilization or destabilization of this stem-loop causes a defect in viral replication [START_REF] Lopez-Lastra | Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors[END_REF].

A.2. The U5 region

The U5 region is composed of 83 nucleotides and is the first part of the genomic RNA to be retro-transcribed. A region of 18 nucleotides called PBS is present immediately at the 3' end of the U5 region that specifically binds to tRNA Lys3 .

A.3. The dimerization / packaging domain

This domain is important for dimerization of the genomic RNA and its selection in the packaging. It also contains the SD (splice donor site) for the splicing of mRNA. In HIV-1, this domain includes four stem-loops SL1 to SL4 [START_REF] Harrison | The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure[END_REF][START_REF] Clever | Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation[END_REF].

The stem-loop SL1 displays a GC-rich 6-nt palindromic sequence at the top of its loop called the dimer initiation signal or DIS [START_REF] Laughrea | A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA[END_REF][START_REF] Paillart | Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA[END_REF][START_REF] Skripkin | Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro[END_REF][START_REF] Muriaux | Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5' leader region is evidenced by an antisense oligonucleotide[END_REF]and is essential for RNA dimerization [START_REF] Skripkin | Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro[END_REF][START_REF] Paillart | Dimerization of retroviral genomic RNAs: structural and functional implications[END_REF] as well as for RNA partner selection in vivo [START_REF] Chin | Identification of a major restriction in HIV-1 intersubtype recombination[END_REF][START_REF] Moore | Dimer initiation signal of human immunodeficiency virus type 1: its role in partner selection during RNA copackaging and its effects on recombination[END_REF][START_REF] Chen | High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis[END_REF]). The autocomplementary nature of the DIS supports the initiation of Watson-Crick base pairing between the two HIV-1 RNA molecules and thus generating a "kissing loop" [START_REF] Clever | Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA[END_REF][START_REF] Paillart | Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA[END_REF][START_REF] Kieken | A new NMR solution structure of the SL1 HIV-1Lai loop-loop dimer[END_REF]) structure. The most common DIS sequence is GCGCGC in subtype B and GUGCAC in subtype A, C and G of HIV-1 strains.

Furthermore, the SL2 loop containing the SD (splicing domain), a motif of nine nucleotides, CUG-GUGAGU, where the dash between nucleotides represent the cleavage site, which is considered as major splice donor site (O'Reilly, [START_REF] O'reilly | Two strong 5' splice sites and competing, suboptimal 3' splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA[END_REF]. It is located in the upper part of a short stem-loop. This means that all HIV-1 transcripts are cleaved at this point by the spliceosome except the full-length RNA. The SL3 loop also called psi (), which is the major site of packaging, has been considered for a long time the only essential loop for packaging [START_REF] Lever | Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions[END_REF][START_REF] Kuzembayeva | Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles[END_REF]. More recent studies have shown that several sequences upstream of SL3, including the SL1 loop, were also essential for a specific encapsidation of genomic RNA [START_REF] Ekram | Specific recognition of the HIV-1 genomic RNA by the Gag precursor[END_REF]. Finally, SL4 loop contains the AUG start codon of Gag [START_REF] Nikolaitchik | Dimeric RNA recognition regulates HIV-1 genome packaging[END_REF].

A.4. The U3 region

The U3 region at 3' end composed of approximately 450 nucleotides and contains the signals required for the regulation of transcription of proviral DNA into viral RNA with the help of transcription machinery of the host cell [START_REF] Gaynor | Cellular transcription factors involved in the regulation of HIV-1 gene expression[END_REF].

There are other non-coding regions within the viral genome:

The PPT (Poly purine Tract) and PPTC (Poly purine Tract central) are purine-rich domains of the RNA genome. The 3' PPT is located immediately upstream of U3 sequence of the 3' LTR of the proviral genome, while the PPTC is located in the open reading frame of the pol gene [START_REF] Charneau | A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication[END_REF]). The RNase H activity of reverse transcriptase degrades the viral genomic RNA during the synthesis of double-stranded DNA, with the exception of two purine rich sequences which serve as primers for the synthesis of the (+) strand DNA.

RRE (Rev Responsive Element

) is composed of 351 nucleotides, consists of several sequences of stem-loops and is located in the env gene. It interacts with the Rev protein of HIV-1, which facilitates nuclear export of non-or semi-spliced viral transcripts [START_REF] Felber | rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA[END_REF]).

B. The coding region

The coding region of HIV-1 is much more complex than in other retroviruses. In addition to the classic three genes gag, pol and env, it encodes for other viral proteins like Tat, Rev, Nef, Vif, Vpr and Vpu (Figure 8).

Viral protein synthesis

Eukaryotic translation begins with the recruitment of the small ribosomal subunit (40S subunit) on the mRNA and its movement until it reaches and localizes the initiation codon (the initiation step) in a favorable Kosak context (A/G)CCAUG. Next step corresponds to the elongation phase where the ribosome decodes the mRNA and links residues in order to get the protein translation.

This elongation stops when the ribosome reads one of three possible termination codons (UGA, UAA or UAG) [START_REF] Alkalaeva | In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3[END_REF]. This final step corresponds to the ribosome recycling for the next round of translation [START_REF] Pisarev | Recycling of eukaryotic posttermination ribosomal complexes[END_REF].

As for cellular mRNA, viral RNAs are capped at their 5' extremity and polyadenylated at their 3' end. These two phenomenons are required for efficient mRNA translation initiation by the 5' cap-dependent initiation mechanism. Translation starts with interaction of eIF2-tRNA Met with the 43S pre-initiation complex (40S ribosome subunit + eIFs). This complex later interacts with the 5'-capped RNA via eIF4F. This latter factor is a DEAD-box RNA helicase that unwinds RNA structures of the 5'UTR, initiating ribosomal scanning till the AUG codon. The 60S large ribosomal subunit can then join to the 40S ribosomal subunit and thus assembling an 80S ribosome. This 80S complex is competent for the elongation step [START_REF] Pestova | The joining of ribosomal subunits in eukaryotes requires eIF5B[END_REF].

Nevertheless, for viruses and in particular for HIV, the limiting process during its RNA translation is its folded structure and the length of 5'UTR. To overcome this difficulty, internal initiation takes place at the level of the internal ribosome entry site (IRES)-mediated translation initiation site [START_REF] Balvay | Structural and functional diversity of viral IRESes[END_REF]. In this case, the 43S pre-initiation complex containing eIF4G and eIF4A binds directly into internal stem loop initiating the scanning to the initiation codon. All viral proteins are synthesized on free polysomes in the cytoplasm except for the precursor Env p160, which is synthesized in the rough endoplasmic reticulum (RER).

Viral structural proteins

A. Gag protein

Gag protein will be described in later sections of thesis.

B. The viral envelope protein (Env)

The Env protein is expressed as the precursor gp160 from a bi-cistronic mono-spliced mRNA.

Like all surface glycoproteins, Env is synthesized in the RER and migrates through the Golgi apparatus where it undergoes glycosylation. Subsequent maturation processing yields the gp120

surface subunit [SU] and gp41 trans-membranous subunit [TM] [START_REF] Checkley | HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation[END_REF]).

The gp120 exists as a trimer located on the surface of the infected cells and of the virions, where it is linked non-covalently to the gp41 (Figure 9).The gp120 harbors five hypervariable regions, V1-V5 interspersed with five constant regions (C1 to C5). On the other hand, gp41 is composed of an N-terminal ectodomain, which contains the fusion peptide, a trans-membrane domain, and a C-terminal intravirion segment that interacts with MA (Figure 9) [START_REF] Checkley | HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation[END_REF].

The viral enzymes

The viral protease

It is a member of the aspartyl protease family, is expressed as a Gag-Pol fusion protein and is released by an autocatalytic mechanism. HIV-1 protease activity is required for cleavage of the

The viral reverse transcriptase (RT)

This enzyme is encoded by the pol gene, and is initially packaged into virions as a Gag-Pol precursor [START_REF] Herschhorn | Retroviral reverse transcriptases[END_REF]. Its proteolytic cleavage primarily produces a homodimer of two p66 molecules each containing both a polymerase and an RNase H domain. The RNase H domain is subsequently removed by proteolysis from one of the subunits giving the mature p51/p66 RT heterodimer. The polymerase domain is composed of four subdomains known as the "fingers'', "palm'', "thumb'', and "connection" which connects the polymerase and RNase H domains (Figure 11). RT has RNA-dependent and DNA-dependent polymerase activities and

Protease active site

Substrate binding pocket Flap

The viral integrase (IN)

HIV integrase mediates the insertion of the HIV proviral DNA into the genomic DNA and is a 32 kDa protein product derived from the C-terminal portion of the pol gene [START_REF] Ciuffi | Retroviral DNA integration: HIV and the role of LEDGF/p75[END_REF]. The three-dimensional structure of the full-length HIV-1 integrase has been resolved by means of X-ray crystallography (Figure 12).The structure of IN contains three canonical domains connected by flexible linkers: an N-terminal HH-CC zinc-binding domain, a central catalytic core domain and a C-terminal DNA-binding domain [START_REF] Karki | Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design[END_REF][START_REF] Cherepanov | Structural insights into the retroviral DNA integration apparatus[END_REF]. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. Among them, it has been shown a stable and functional complex between the wild-type full-length integrase and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone [START_REF] Michel | Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor[END_REF]. By using mass spectrometry and cryo-EM it was shown that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules [START_REF] Michel | Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor[END_REF]. One integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer, shedding the light on the integration mechanism.

Viral regulatory and auxiliary proteins

The trans-activator of the transcription (Tat)

Tat is an RNA binding protein that acts as a trans activator of viral transcription [START_REF] Pugliese | A review of HIV-1 Tat protein biological effects[END_REF] and can be found in two forms of 72 or 101 amino acids from two coding exon. The structure of Tat consists of four domains: the cysteine-rich segment, the core, a basic segment rich in arginine responsible for TAR recognition and binding, and a glutamine rich segment [START_REF] Tahirov | Crystal structure of HIV-1 Tat complexed with human P-TEFb[END_REF]) (Figure 13). It is responsible for enhancing expression of some cellular factors including tumor necrosis factor beta (TNF-β), and transforming growth factor beta. On the other hand, it acts to suppress the expression of other cellular genes including bcl-2 and the chemokine, MIP-1α [START_REF] Pugliese | A review of HIV-1 Tat protein biological effects[END_REF]. Cdk9 is light orange, Cyclin T1 is pale green and Tat is magenta. The side chains of the Cdk9-interacting residues of Tat, Cys261 of Cyclin T1, and ATP analog are drawn as sticks, and the zinc and magnesium atoms are drawn as cyan and light blue spheres, respectively. The dashed lines represent the missing link between Lys88 and Gly35 of Cdk9 and between Leu252 and Cys261 of Cyclin T1 [START_REF] Tahirov | Crystal structure of HIV-1 Tat complexed with human P-TEFb[END_REF] Furthermore, as far its functions are concern, Tat is considered to stimulate the RTion reaction through its ability to chaperone the annealing of the primer tRNA onto the viral RNA [START_REF] Kameoka | The Tat protein of human immunodeficiency virus type 1 (HIV-1) can promote placement of tRNA primer onto viral RNA and suppress later DNA polymerization in HIV-1 reverse transcription[END_REF] as well as the first cDNA strand transfer [START_REF] Guo | Suppression of an intrinsic strand transfer activity of HIV-1 Tat protein by its second-exon sequences[END_REF]. It has been reported that Tat exhibits potent nucleic acid chaperone activities similar to those of the HIV-1 nucleocapsid protein NCp7 [START_REF] Godet | Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1[END_REF]) and of the Flaviviridae core protein [START_REF] Ivanyi-Nagy | RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae[END_REF], which is mediated by the basic 44-61 peptide sequence [START_REF] Kuciak | The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro[END_REF][START_REF] Boudier | The mechanism of HIV-1 Tat-directed nucleic acid annealing supports its role in reverse transcription[END_REF]).

The viral regulatory protein (Rev)

The protein Rev is a 13-kDa (116 residues) sequence-specific RNA binding protein and is trafficked into the nucleus after its synthesis in the cytoplasm. It is involved in the nuclear export of HIV unspliced and singly-spliced mRNAs by binding to the RRE [START_REF] Suhasini | Cellular proteins and HIV-1 Rev function[END_REF]. This Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs up to 200-fold, irrespective of whether RNAs have been spliced or not [START_REF] Grewe | The HIV-1 Rev protein enhances encapsidation of unspliced and spliced, RRE-containing lentiviral vector RNA[END_REF]. By using X-ray crystallography, three-dimensional structures of HIV-1 Rev and the multimeric RNA-protein complex that forms on the RRE has already been revealed (Fig. 14). It shows that Rev can form homo-tetramers and contains three functional

The viral negative factor (Nef)

Nef is a 27-kDa protein and undergoes post-translational modification by phosphorylation and N-terminal myristoylation (Figure 15). It is encoded by a single exon that extends into the 3' LTR of a multispliced mRNA. At later stages of viral life cycle, it is cleaved by the viral protease after packaging during virion maturation, however the relevance of this event is not clear [START_REF] Turner | Structural biology of HIV[END_REF].

Nef plays a major role in the evolution of viral infection towards an immunodeficiency syndrome. It promotes virus dissemination by releasing virus from infect cells and by increasing cell/cell transfer of virions. However, it takes part in avoiding super-infection by downregulating HIV-1 receptors like CD4 from plasma membrane [START_REF] Laguette | Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins[END_REF]. Nef (orange) binds to the alpha subunits (cyan) and sigma 2 (magenta) of AP-2. (Ren X 2014).

The viral protein R (Vpr)

This protein is composed of 96 amino acids and about 12.7 kDa [START_REF] Fritz | HIV-1 Viral Protein R : from structure to function[END_REF]. Vpr performs its functions over many steps in the retroviral cycle, particularly during the early phase where it may be involved in the fidelity of reverse transcription. Furthermore, Vpr is part of PIC and participate in the transit of the proviral DNA into the nucleus through its two NLS [START_REF] Guenzel | HIV-1 Vpr-a still "enigmatic multitasker[END_REF]. Although the nuclear import of viral DNA process is still uncertain, it appears that Vpr is involved, especially in quiescent cells or macrophages cells latent kind, maintaining the PIC near nuclear pores (NPC) through its interaction with nucleoporin hCG1 [START_REF] Le Rouzic | Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1[END_REF]. Furthermore, it has recently been shown that Vpr is capable of reorganizing dsDNA more compactly [START_REF] Lyonnais | A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA[END_REF]. Finally, Vpr also has the property to destabilize the nuclear membranes in the cell medium [START_REF] Piller | The amino-terminal region of Vpr from human immunodeficiency virus type 1 forms ion channels and kills neurons[END_REF][START_REF] De Noronha | Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr[END_REF][START_REF] Brugger | The HIV lipidome: a raft with an unusual composition[END_REF], suggesting that Vpr may participate in passive PIC transit through the disturbed areas of the nuclear membrane (Figure 16).

NMR studies were performed to obtain the structure of Vpr and its various fragments, in different solvent condition, due to the aggregation prone of the protein and it was observed that the protein contains three helical domains (Figure 16). The first α-helix D17-H33, seems to be responsible for nuclear import and virion incorporation, the second α-helix, W38-Y50 appears to be responsible for the oligomerization of Vpr and the third α-helix, W54-R77, derives the nuclear transport and amplification of viral replication in non-dividing cells [START_REF] Morellet | Structure-function relationship of Vpr: biological implications[END_REF]). The protein adopts the same secondary and three-dimensional conformation, a similar orientation of the three amphipathic α-helices folding around a hydrophobic core, in pure H 2 O as in presence of CH 3 CN. Structure revealed that the three successive α-helices are mutually oriented to promote lipophilic interactions between the hydrophobic amino acid side chains of helix I, helix II and helix III [START_REF] Morellet | NMR structure of the HIV-1 regulatory protein VPR[END_REF].

Figure 16: Three-dimensional structure of the HIV-1 Vpr protein [START_REF] Morellet | NMR structure of the HIV-1 regulatory protein VPR[END_REF]. Alpha helices are colored in pink, orange and blue. The Trp54 residue responsible for interactions with cellular proteins is also highlighted.

Furthermore, it is considered as a regulatory protein that is incorporated in particles along the budding. This incorporation depends on NCp7 and p6 domains of Gag (de [START_REF] De Rocquigny | The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions with the viral regulatory protein Vpr[END_REF][START_REF] Bachand | Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor[END_REF]. Interestingly, this incorporation is highly sensitive to the Vpr oligomerisation. In fact, Vpr was shown to form dimers and trimers in the cytoplasm and in the nucleus (Fritz, Didier et al. 2008) that are essential for Gag recognition [START_REF] Fritz | HIV-1 Viral Protein R : from structure to function[END_REF]Fritz, Dujardin et al. 2010).

Among the range of functions of the Vpr protein, the Vpr-dependent G2 arrest activity was extensively explored since its discovery [START_REF] He | Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity[END_REF][START_REF] Jowett | The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle[END_REF][START_REF] Refaeli | The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product[END_REF][START_REF] Rogel | The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection[END_REF]. HIV-1 LTR was shown more active in the G2 phase, implying that the G2 arrest may confer a favorable cellular environment for efficient transcription of HIV-1 [START_REF] Goh | HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo[END_REF]. The mechanism whereby Vpr indices G2 arrest involved many different pathways such as p34cdc2/cyclinB [START_REF] Goh | HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo[END_REF], formation of a complex with p53 and sp1 transcription factor [START_REF] Wang | Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and trans-activation of viral long terminal repeat[END_REF][START_REF] Sawaya | Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription[END_REF]. More recently, the interaction of Vpr with the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) has been shown crucial for the G2arrest activity [START_REF] Laguette | Premature activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from innate immune sensing[END_REF]).

The viral protein U (Vpu)

It is a 16-kDa protein found as an integral membrane phosphoprotein primarily localized in the internal membranes of the cell [START_REF] Maldarelli | Human immunodeficiency virus type 1 Vpu protein is an oligomeric type I integral membrane protein[END_REF]. The structure of Vpu consists of a hydrophobic NTD that functions both as signal and membrane anchor peptide, and a hydrophilic CTD, which protrudes into the cytoplasm (Figure 17). However, the 3-D structure of the fulllength HIV-1 Vpu has not yet been determined [START_REF] Dube | Modulation of HIV-1-host interaction: role of the Vpu accessory protein[END_REF]. This protein displays two major functions during HIV-1 replication cycle. Firstly, Vpu targets the newly synthesized CD4 receptor to the proteasomal degradation [START_REF] Willey | Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4[END_REF]). Secondly, it favors the egress of viral particles from most human cell types through counteracting the inhibitory effect of BST2 [START_REF] Neil | Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu[END_REF][START_REF] Van Damme | The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein[END_REF]). More recently, Vpu was shown to down-regulate cell surface expression of two additional mediators of the immune response: the lipid-antigen presenting protein CD1d expressed by antigen-presenting cells [START_REF] Moll | Inhibition of lipid antigen presentation in dendritic cells by HIV-1 Vpu interference with CD1d recycling from endosomal compartments[END_REF]) and the natural killer cells ligand NTB-A [START_REF] Shah | Degranulation of natural killer cells following interaction with HIV-1-infected cells is hindered by downmodulation of NTB-A by Vpu[END_REF]).

The viral infectivity factor (Vif)

It is a 32 kDa basic protein that being produced during the late phase of the viral life cycle [START_REF] Henriet | Tumultuous relationship between the human immunodeficiency virus type 1 viral infectivity factor (Vif) and the human APOBEC-3G and APOBEC-3F restriction factors[END_REF]. About 60-100 molecules per virion are packaged via an interaction with viral gRNA and the precursors Gag/Gag-Pol. It is found essential for the viral pathogenesis and replication in peripheral blood lymphocytes, macrophages, and certain nonpermissive cell lines [START_REF] Henriet | Tumultuous relationship between the human immunodeficiency virus type 1 viral infectivity factor (Vif) and the human APOBEC-3G and APOBEC-3F restriction factors[END_REF]. A three-dimensional structural model for Vif is available, that was constructed by a comparative modeling based on two templates (VHL and NarL) used to construct its C-terminal and N-terminal domains (Figure 18) [START_REF] Lv | Three-dimensional structure of HIV-1 VIF constructed by comparative modeling and the function characterization analyzed by molecular dynamics simulation[END_REF]). In vitro, Vif was shown to oligomerize to form dimers, trimmers and tetramers [START_REF] Yang | The multimerization of human immunodeficiency virus type I Vif protein: a requirement for Vif function in the viral life cycle[END_REF][START_REF] Auclair | Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity[END_REF], possibly mediated by the region from positions 151 to 164 amino acids [START_REF] Yang | The multimerization of human immunodeficiency virus type I Vif protein: a requirement for Vif function in the viral life cycle[END_REF][START_REF] Yang | Potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type 1 (HIV-1) Vif proteins[END_REF].

Initially, Vif has been shown to interact with Gag in vitro as well as in infected cells [START_REF] Bouyac | Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor[END_REF]. Later on, the interaction was investigated further by using two-hybrid assay and it was observed that Vif can interact with nucleocapsid proteins of Gag. It was revealed that amino-terminal (1-22 aa) and central (77-100 aa) regions of Vif are essential for its interaction with Gag. On the other hand, Gag requires a 35-amino acid region of the protein bridging the MA(p17)-CA(p24) junction for its interaction with Vif [START_REF] Syed | Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55(GAG)) of the virion nucleocapsid proteins[END_REF]. In addition, oligomerization of Vif is not critical for Gag recognition, but the interaction between these two proteins leads to the redistribution of Vif protein at the plasma membrane, which likely mediates Vif incorporation into nascent virions [START_REF] Batisse | APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells[END_REF][START_REF] Ludovic Richert | Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy[END_REF]. The main function of Vif concerns its interaction with APOBEC3 family to an E3 ubiquitin ligase complex for polyubiquitylation and proteasomal degradation. During this process Vif adopts a noval structural fold that determines Vif stability and interaction with cellular proteins and motifs driving Vif homodimerization. Both of these functions are essential in Vif functionality and HIV-infection [START_REF] Batisse | APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells[END_REF][START_REF] Salter | Structural insights for HIV-1 therapeutic strategies targeting Vif[END_REF]).

Assembly of the viral particles

Gag protein:

Gag is encoded in the Gag-pol gene and is 55 kDa polyprotein. A translational frame-shift in the 3′ region of Gag regulates the expression of either Gag or the fusion protein Gag-Pol [START_REF] Karn | Transcriptional and posttranscriptional regulation of HIV-1 gene expression[END_REF]. It has been shown that Gag alone is able to mediate the assembly of immature VLPs.

Gag consists of a series of globular, α-helical domains connected by flexible linkers. It contains five proteolytic sites that are cleaved by the viral PR during maturation to generate three proteins: Matrix (MA), Capsid (CA) and Nucleocapsid (NC) and three peptides: SP1, SP2 and p6 (Figure 19). Processing of Gag leads to the nucleocapsid condensation. After binding to nucleic acids, NCp15, a proteolytic intermediate of NC, was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and SP1. This allows NC co-aggregation with its single-stranded nucleic acid substrate. Thus, the interchangeable roles of HIV-PR and RT, by controlling the NC architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodeling process. This process is among the common features developed by mobile genetic elements to ensure successful replication [START_REF] Mirambeau | HIV-1 protease and reverse transcriptase control the architecture of their nucleocapsid partner[END_REF]. By using Small-angle scattering, Gag has been found highly flexible protein, with the domains moving freely around the flexible linkers [START_REF] Datta | Conformation of the HIV-1 Gag protein in solution[END_REF]). This flexibility is particularly prominent for the linker between MA and CA. This flexibility makes the Gag polyprotein difficult to study in its entirety using structural biology methods, still a good amount of knowledge has been retrieved by studying individual constituent domains in isolation.

(A)

Matrix protein: architect of Membrane Targeting

132 amino acid long structural matrix protein is derived from the N-terminal myristoylated end of the Gag (MAp15) (Figure 21B) and remains attached with the inner surface of the viral envelope in the mature particle [START_REF] Chukkapalli | Molecular determinants that regulate plasma membrane association of HIV-1 Gag[END_REF]. HIV-1 MA folds into a compact core domain, consisting largely of 5 α-helices, two short 3 α-helical stretches and a threestranded mixed β-sheet (Figure 21A) [START_REF] Verli | Molecular dynamics analysis of HIV-1 matrix protein: clarifying differences between crystallographic and solution structures[END_REF]). This protein exists in a monomeric state in solution, but crystallizes as trimmers that can be correlated with the formation of higher-order MA structures required for its function in the viral life cycle.

As myristoylation is essential for efficient membrane binding and assembly, the N-terminus of MA domain plays a major role in Gag targeting to the cytoplasmic leaflet of the PM [START_REF] Bryant | Myristoylation-dependent replication and assembly of human immunodeficiency virus 1[END_REF][START_REF] Yuan | Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor[END_REF][START_REF] Freed | Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production[END_REF][START_REF] Zhou | Identification of a membrane-binding domain within the aminoterminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids[END_REF][START_REF] Saad | Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly[END_REF]). The N-terminal myristic acid moiety [START_REF] Bryant | Myristoylation-dependent replication and assembly of human immunodeficiency virus 1[END_REF][START_REF] Saad | Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly[END_REF]) and a highly basic region on the surface of MA (residues 16-31) [START_REF] Yuan | Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor[END_REF][START_REF] Zhou | Identification of a membrane-binding domain within the aminoterminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids[END_REF]) are required for membrane association [START_REF] Hermida-Matsumoto | Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging[END_REF][START_REF] Ono | Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly[END_REF]. The myristoyl moiety of MA is thought to exist in either an exposed or a sequestered conformation which is in analogy to other myristoylated proteins [START_REF] Tang | Entropic switch regulates myristate exposure in the HIV-1 matrix protein[END_REF]). It has been suggested that most of myristoylated proteins regulate membrane association through a myristoyl switch mechanism, in case of Gag, a concentrationdependent oligomerisation triggered the myristoyl switch and facilitate membrane binding [START_REF] Tang | Entropic switch regulates myristate exposure in the HIV-1 matrix protein[END_REF]). This allosteric conformational change that exposes the myristoyl moiety of Gag due to the interaction of MA and PI(4,5)P 2 , [START_REF] Saad | Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly[END_REF]. PI(4,5)P 2 is a inositol phospholipid found on the cytosolic leaflet of the PM and is a major determinant for PM targeting by a number of cytoplasmic peripheral PM proteins [START_REF] Behnia | Organelle identity and the signposts for membrane traffic[END_REF]. PI(4,5)P 2 embedded in the inner leaflet of the PM lipid bilayer is shown with its (orange) and (yellow) acyl chains in the lipid bilayer. 2: binding between MA and PI(4,5)P 2 leads to the flipping out of the myristate moiety into an exposed conformation with its subsequent insertion into the lipid bilayer, and the extrusion of the 2′acyl chain from the lipid bilayer and its sequestration by MA. 3: during the formation of the complex between HIV-1 MA and PI(4,5)P 2 , the 2′-unsaturated acyl chain of PI(4,5)P 2 (yellow) binds to the hydrophobic cleft in MA and the myristyl group (green) of MA inserts into the lipid bilayer [START_REF] Balasubramaniam | New insights into HIV assembly and trafficking[END_REF].

The MA domain is highly basic, harbors an NTD HBR (Highly Basic Region) and could function by interacting directly with the viral RNA as shown by in vitro nucleic acid binding assays [START_REF] Steeg | RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses[END_REF][START_REF] Purohit | Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection[END_REF][START_REF] Cai | Structural basis of the association of HIV-1 matrix protein with DNA[END_REF][START_REF] Alfadhli | HIV-1 matrix protein binding to RNA[END_REF]. Virions lacking GagNC were shown to incorporated significant amount of viral RNA suggesting that MA was involved in the recognition of RNA (Ott, Coren et al. 2005).

Capsid protein: Major Structural Element

The CA protein is main brick of architect that is required for Gag-Gag interactions in the immature shell as well as in the mature particles and comprises two independent domains (NTD & CTD) separated by a flexible linker (Figure 22).The structure of the individual CA-CTD and CA-NTD has been resolved recently, by using cryoelectron microscopy (cryo-EM) and cryotomography and NMR spectroscopy [START_REF] Briggs | The molecular architecture of HIV[END_REF][START_REF] Yeager | Design of in vitro symmetric complexes and analysis by hybrid methods reveal mechanisms of HIV capsid assembly[END_REF]).

The immature capsid shell is arranged in a lattice formed by Gag hexamers stabilized by a six helix bundle of SP1 peptides and are joined through CTD dimer interface, whereas the residues of the NTD contribute to intra-hexamer and/or inter-hexamer interactions [START_REF] Mateu | The capsid protein of human immunodeficiency virus: intersubunit interactions during virus assembly[END_REF].

Furthermore, mature HIV-1 CA adopts a cone-shaped shell forming a curved hexagonal lattice arranged in a fullerene-type geometry consisting of ~250 hexameric subunits with a 90-100 Å inter subunit spacing (Fig. 21) [START_REF] Briggs | The molecular architecture of HIV[END_REF][START_REF] Yeager | Design of in vitro symmetric complexes and analysis by hybrid methods reveal mechanisms of HIV capsid assembly[END_REF].

The nucleocapsid protein (NCp7)

Main architect in Recognition and Binding of Viral Genome (reviewed in (Darlix, de Rocquigny et al. 2014))

The HIV nucleocapsid (NCp7) protein harbors two conserved "CCHC domains'' (Cys-X2-Cys-X4-His-X4-Cys; where X is a variable amino acid residue) forming the zinc-finger (ZF) motifs (Figure 24B) that contains a zinc ion each and is flanked on either side by linker harboring highly basic residues (RAPRKKG) (Figure 24) (Morellet, Jullian et al. 1992;Muriaux and Darlix 2010;[START_REF] Darlix | Flexible nature and specific functions of the HIV-1 nucleocapsid protein[END_REF]. Enriched literature data showed that the NTD-ZF1 is essential for genome recognition, whereas the CTD-ZF2 would be more involved in viral assembly and the early stages of viral replication (Houzet, Morichaud et al. 2008). GagNC of HIV-1 is essential during many key processes both during the early steps of the viral life cycle as the mature GagNC protein and during the late steps as domain of the precursor Gag [START_REF] Mirambeau | Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function[END_REF]):

i) It helps in the recognition and specific packaging the viral genomic RNA (gRNA) (Later described in details) [START_REF] Darlix | Flexible nature and specific functions of the HIV-1 nucleocapsid protein[END_REF].

ii) it entirely coats and protects the genome forming a compact ribonucleoprotein (RNP) complex in the viral particle maturation, with a very high degree of occupancy (1:5 nt up to 1:1) [START_REF] Darlix | First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses[END_REF] vii) It participates to viral DNA integration as shown by cDNA integration in vitro [START_REF] Carteau | Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro[END_REF]). Additionally, it has been shown that GagNC and Integrase cooperate to ensure protection of the inverted repeat sequences ''ir''at both ends of the LTR [START_REF] Bampi | The chaperoning and assistance roles of the HIV-1 nucleocapsid protein in proviral DNA synthesis and maintenance[END_REF]).

iii) it drives directly or indirectly the incorporation in nascent particles of cellular APOBEC3G [START_REF] Alce | APOBEC3G is incorporated into virus-like particles by a direct interaction with HIV-1 Gag nucleocapsid protein[END_REF], viral protein Vif [START_REF] Huvent | Interaction and co-encapsidation of human immunodeficiency virus type 1 Gag and Vif recombinant proteins[END_REF][START_REF] Bardy | Interaction of human immunodeficiency virus type 1 Vif with Gag and Gag-Pol precursors: co-encapsidation and interference with viral protease-mediated Gag processing[END_REF], and Vpr [START_REF] Bachand | Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor[END_REF]).

iv) it has a RNA chaperon property essential to enhance dimerization of viral RNA [START_REF] Darlix | Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1[END_REF]De Rocquigny, Gabus et al. 1992) and its conversion into a more compact and stable form, a process known as ''RNA maturation'' [START_REF] Feng | HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro[END_REF][START_REF] Muriaux | NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer[END_REF].

Nucleic acid chaperone activity during the replication cycle:

Nucleic acid chaperones have the ability to direct structural rearrangements of secondary structures of nucleic acids (DNA and RNA) via rapid iterative on and off binding to the nuclei acid. Such a binding mode helps nucleic acids to overcome 'kinetically trapped inactive states' in order to rapidly reach the most stable conformation [START_REF] Herschlag | RNA chaperones and the RNA folding problem[END_REF][START_REF] Kovacs | Janus chaperones: assistance of both RNA-and protein-folding by ribosomal proteins[END_REF]). This chaperone activity of nucleic acids is essential during reverse transcription, by inducing hybridization tRNA Lys3 to the viral RNA, by increasing the speed of the reverse transcriptase and activating strongly the transfer of strands [START_REF] Levin | Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism[END_REF]Godet, de Rocquigny et al. 2006;Ramalanjaona, de Rocquigny et al. 2007;[START_REF] Rein | Diverse interactions of retroviral Gag proteins with RNAs[END_REF]Darlix, de Rocquigny et al. 2014). This activity consists of three steps: interaction with target nucleic acids, destabilization of strands and hybridization of complementary strands. The NCp7chaperoned folding of NAs has been characterized by both a NA destabilizing activity and a complementary strand annealing activity (Reviewed in [START_REF] Godet | Biophysical studies of the nucleic acid chaperone properties of the HIV-1 nucleocapsid protein[END_REF]). The destabilizing activity of GagNC appears to be weak and strongly dependent on the NA stability, resulting in a transient melting of 'weak spots' within the NA. The locally transiently-melted areas allow conformational sampling, resulting in the selection of stable conformations. On the other hand, the ability of NCp7 to efficiently screen repulsive charges and aggregate NAs, mainly through its basic residues, strongly enhances the limiting nucleation step during annealing of complimentary NAs. Thus, the destabilizing activity of NCp7 depends essentially on zinc fingers [START_REF] Beltz | Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis[END_REF] while the hybridization and condensation activities depend essentially from basic parts surrounding the CCHC motifs (De Rocquigny, Gabus et al. 1992;[START_REF] Williams | Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching[END_REF]) (Figure 24 B).

Furthermore, Gag protein has also a chaperone activity but that seems less efficient than the one of GagNC [START_REF] Rein | Nucleic acid chaperone activity of retroviral Gag proteins[END_REF][START_REF] Wu | Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications[END_REF]). Interestingly, it has been shown that this chaperone activity was increased with the maturation products of Gag NCp15, NCp9 and NCp7 [START_REF] Wu | Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications[END_REF]Wang, Naiyer et al. 2014).

Conversely, the Gag protein interacts strongly with nucleic acids and this affinity decreases in the different maturation products. This suggests a model in which in the early stages of assembly, Gag protein requires a strong affinity to recognize and interact with the genomic RNA.

Then, during maturation by the viral protease, this affinity decreases in the benefit of the chaperone activity promoting reverse-transcription [START_REF] Mirambeau | Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function[END_REF][START_REF] Godet | Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1[END_REF]).

The protein p6: Recruiter of Cellular Budding Machinery

p6 is a small proline-rich protein of 6 kDa and is located at the C terminus of Gag. It is separated from GagNC by SP2 (Figure 20E). As the N terminus of this domain is encoded by sequences that direct translational frame shift into the overlapping pol gene [START_REF] Klein | Intracellular destinies: degradation, targeting, assembly, and endocytosis of HIV Gag[END_REF]), thus, it is absent from the Gag-Pol precursor. This deletion results in impaired virus release [START_REF] Gottlinger | The HIV-1 assembly machine[END_REF].

The p6 domain contains two highly conserved the P(T/S)AP and LXXLF motifs at its N and C termini, respectively. The NTD, also called assembly or "L" late domain is involved in the viral budding [START_REF] Morita | Retrovirus budding[END_REF][START_REF] Yeager | Design of in vitro symmetric complexes and analysis by hybrid methods reveal mechanisms of HIV capsid assembly[END_REF]. LXXLF is also considered as late domain as well as is needed for the incorporation of Vpr protein into the assembling virions [START_REF] Gottlinger | The HIV-1 assembly machine[END_REF]. Next, this domain is necessary for recruiting the ESCRT-I component TSG101 (Later described in details). Interestingly, these motifs are also found in many viruses such as ebola, RSV, Marburg, Mason-Pfizer monkey virus, bovine leukemia virus, Human T-cell leukemia virus type 1, etc [START_REF] Strack | A role for ubiquitin ligase recruitment in retrovirus release[END_REF][START_REF] Herrington | Viruses and disease: emerging concepts for prevention, diagnosis and treatment[END_REF].

SP1 (p2) & SP2 (p1)

SP1 and SP2 are two small spacer peptides, cleaved during viral particle maturation and separates CA with NC, and NC with p6, respectively (Figure 20). These peptides influence precursor polyprotein processing and viral morphogenesis which is required to produce fully infectious virions [START_REF] Gottlinger | The HIV-1 assembly machine[END_REF][START_REF] Klein | Intracellular destinies: degradation, targeting, assembly, and endocytosis of HIV Gag[END_REF]). However, their precise functions are controversial. With a possibility that SP1 can function as a molecular switch region that controls the transition from sphere to cone, it also contributes with CA to the interface of Gag-Gag multimerization [START_REF] Morellet | Structure of the complex between the HIV-1 nucleocapsid protein NCp7 and the single-stranded pentanucleotide d(ACGCC)[END_REF][START_REF] Morellet | Helical structure determined by NMR of the HIV-1 (345-392)Gag sequence, surrounding p2: implications for particle assembly and RNA packaging[END_REF][START_REF] Datta | On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch?[END_REF]).

Gag assembly and trafficking

A generally accepted model for HIV-1 assembly stipulates that the genomic RNA acts as a scaffolding platform onto which Gag molecules bind via GagNC, then kick-starting assembly by CA-CA interaction (Ott, Coren et al. 2009;[START_REF] Kutluay | Analysis of the initiating events in HIV-1 particle assembly and genome packaging[END_REF][START_REF] Fogarty | Characterization of cytoplasmic Gag-gag interactions by dual-color z-scan fluorescence fluctuation spectroscopy[END_REF][START_REF] O'carroll | Elements in HIV-1 Gag contributing to virus particle assembly[END_REF]. This, in turn causes the binding of N-terminal myristyl switch of Gag MA or binding of the RNA-bound Gag oligomers [START_REF] Lindwasser | Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains[END_REF][START_REF] Ono | Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane[END_REF][START_REF] Saad | Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly[END_REF][START_REF] Li | Myristoylation is required for human immunodeficiency virus type 1 Gag-Gag multimerization in mammalian cells[END_REF]) to either the plasma membrane (PM) [START_REF] Neil | HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane[END_REF]Welsch, Keppler et al. 2007;Jouvenet, Bieniasz et al. 2008;Ivanchenko, Godinez et al. 2009) or to endosomes (Grigorov, Arcanger et al. 2006;Perlman and Resh 2006;[START_REF] Joshi | Evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment[END_REF][START_REF] Lehmann | Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning[END_REF]).

Gag alone is required for the viral assembly, this assembly occurring either at the level of the PM [START_REF] Ono | HIV-1 Assembly at the Plasma Membrane: Gag Trafficking and Localization[END_REF], or in intracellular vesicles like multivesicular bodies (MVB) [START_REF] Nydegger | HIV-1 egress is gated through late endosomal membranes[END_REF][START_REF] Dong | AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly[END_REF]Grigorov, Arcanger et al. 2006;Perlman and Resh 2006). It was observed that both mechanisms of Gag assembly and trafficking are cell line dependent, indicating that the cellular context has a major influence on the mechanism of Gag assembly and trafficking (reviewed in [START_REF] Balasubramaniam | New insights into HIV assembly and trafficking[END_REF]).

Mutational analysis revealed that only MA, CA and NC are required for Gag assembly while the p6 domain was shown more essential for budding and release. MA governs the targeting to the membrane either by its myristate and HBR, CA contains residues critical for Gag-Gag interaction and GagNC is responsible for viral genomic RNA packaging as well as non-specific interaction of RNA. In line with this, replacement of the HIV-1 GagNC domain by a leucine zipper protein dimerization (or trimerization) domain that allows Gag-Gag contact does not impair virus assembly and release, albeit with no detectable viral RNA.

This retroviral Gag oligomerization and trafficking were obtained by mainly using molecular imaging for recent review [START_REF] Chojnacki | Investigation of HIV-1 assembly and release using modern fluorescence imaging techniques[END_REF][START_REF] De Rocquigny | HIV-1 Gag Directed Assembly of Retroviral 1 Particles Investigated by Quantitative 2 Fluorescence Imaging[END_REF]de Rocquigny, El Meshri et al. 2014). Gag-Gag interactions at the level of the PM were first visualized by two photon imaging, using Rous sarcoma virus (RSV) followed by studies onto HIV-1 Gag. Therefore, a fluorescent reporter was localized to the C terminus of Gag (gives rise to abnormal immature virus structures (Larson, Johnson et al. 2005)) or between MA and CA. In this latter case, when co-expressed with wild-type Gag, infectious virus particles are assembled with apparently normal morphology (Muller, Daecke et al. 2004;Hubner, Chen et al. 2007). As far as HIV-1 is concern, Gag-Gag interaction was confirmed using an approach similar to FRET,

where Gag fluorescent chimeras were expressed in Mel JuSo cells (human melanoma cell line) (Derdowski, Ding et al. 2004), HeLa cells (Jouvenet, Bieniasz et al. 2008;Hogue, Hoppe et al. 2009) and epithelial 293T cells (Hubner, Chen et al. 2007). Alternatively, another strategy was also used by following interactions with FLIM. The FLIM technique measures the fluorescence decay of a chromophore at each pixel or group of pixels of the cell, allowing determining the fluorescence lifetime () (see Mat and Met for details on FRET FLIM). This strategy was applied to follow the fluorescence lifetime of FlAsH linked to HIV-1 Gag-TC (Fritz, Dujardin et al. 2010). Gag oligomerization was followed by the decrease in the fluorescence lifetime of FlAsH, due to its self-quenching and/or exciton coupling [START_REF] Lakowicz | Fluorescence spectroscopic investigations of the dynamic properties of proteins, membranes and nucleic acids[END_REF]Krishnamoorthy, Duportail et al. 2002). The studies were performed by using Hela cells, transfected by a plasmid encoding for Gag-TC, (see in material and methods) that showed Gag accumulation at the level of the PM and the dropping of FlAsH lifetime to 2.57 ns (Figure 26, panel 2, blue pseudo color), as compared to 3.52 ns of the control experiments. Similar FRET studies were performed to determined that SP1 [START_REF] Krausslich | The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity[END_REF][START_REF] Accola | Isolation of human immunodeficiency virus type 1 cores: retention of Vpr in the absence of p6(gag)[END_REF], has a severe impact on Gag-Gag FRET [START_REF] Liang | A structurally disordered region at the C terminus of capsid plays essential roles in multimerization and membrane binding of the gag protein of human immunodeficiency virus type 1[END_REF][START_REF] Von Schwedler | Functional surfaces of the human immunodeficiency virus type 1 capsid protein[END_REF]Hogue, Hoppe et al. 2009), while membrane-defective Gag can still oligomerize in the cytoplasm [START_REF] Lee | Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes[END_REF][START_REF] Morikawa | Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly[END_REF]Derdowski, Ding et al. 2004;[START_REF] Li | Myristoylation is required for human immunodeficiency virus type 1 Gag-Gag multimerization in mammalian cells[END_REF]). The use of this technique to determine the role of GagNC in Gag-Gag interaction was developed during my thesis but with Gag-eGFP and Gag-mCherry and is presented in the results.

Next, investigations were performed to determine the place of Gag oligomer formation inside cell. Furthermore, to address the question of whether VLPs detected at the surface of HeLa cells resulted from Gag clusters arriving from the cytoplasm or from Gag molecules assembling at the PM [START_REF] Ruthardt | Single-particle tracking as a quantitative microscopy-based approach to unravel cell entry mechanisms of viruses and pharmaceutical nanoparticles[END_REF], Total Internal Reflection Fluorescence (TIRF) and wide-field

52

(WF) microscopy were used (Ivanchenko, Godinez et al. 2009). WF images were recorded in the green channel with an excitation of the photoconverted Gag-EOS at 488 nm, while TIRF images were recorded in the red channel with an excitation at 516 nm, after a pulse at 405 nm (Figure 26). By following, the emission of Gag-EOS in green and red channel, it was concluded that VLP formation at the surface of HeLa cells likely resulted from the accumulation of Gag oligomers coming from the cytoplasm. Finally, recent data using TIRF microscopy approach have allowed to track the appearance/disappearance of the viral particle into the plasma membrane. These data, obtained from HeLa cells, show that a first population of viral particles appear/disappear with very short time (5-10 sec.) and their co-localisation with the endosomal marker CD63 suggest their internalization by endocytosis (Jouvenet, Bieniasz et al. 2008). A second population is described with appearance of Gag kinetics about ten minutes followed by a longer period of about 25 minutes which can correspond to either an assembly at the PM from a cytosolic fraction of Gag or re-internalization of Gag in the cytoplasm (Jouvenet, Bieniasz et al. 2008;Ivanchenko, Godinez et al. 2009). Thus, the step of localisation of Gag to the membrane and budding could be influenced by a balance between the extracellular release of Gag as a viral particle and its internalization into the cell by endocytosis.

Additionally, in in vitro conditions, MA and NC domains of monomeric Gag bind to gRNA and adopts a U shape conformation that brings these two domains closer [START_REF] Datta | Conformation of the HIV-1 Gag protein in solution[END_REF]).

To achieve the process of condensation, then MA recruits and interacts tightly with PI( 4 However, it remains to be determined if this U-structure is also found in cells as well mechanism involved behind this phenomenon. There is a possibility that we may differ with the in vitro findings as in cells Gag can co-opts a host protein that either prevents Gag from adopting the folded-over form, or facilitates conversion from the folded-over to the extended form.

In addition to this, as conformational studies have only been performed with myr(-)GagΔp6, the effect of highly unstructured p6 domain in these folded-over conformations needs to be tested.

More recently, Mckinstry et al., have managed to purify full length Gag (myr(-)Gag) and could be crucial in answering these questions [START_REF] Mckinstry | Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli[END_REF]).

Gag-RNA specific recognition for packaging

Schematically, two types of Gag-RNA interactions are described. Initially, non-specific interactions with either cellular RNA or genomic RNA; this interaction drives Gag oligomerisation during the assembly and also allows a RNA wrapping to protect the viral genome from protease attack. Secondly, a more specific interaction has been described between the 5'UTR and the GagNC domain of the Gag polyprotein. Extensive studies delineate various positions of the UTR recognized by GagNC such as TAR [START_REF] Kanevsky | Specific interactions between HIV-1 nucleocapsid protein and the TAR element[END_REF]), SL1, SL2 and SL3 [START_REF] Darlix | Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1[END_REF]Clever, Sassetti et al. 1995;[START_REF] Amarasinghe | NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform[END_REF][START_REF] Houzet | HIV controls the selective packaging of genomic, spliced viral and cellular RNAs into virions through different mechanisms[END_REF]).

Despite the abundance of studies, still there exist many unsolved issues related to the molecular interactions responsible for selective gRNA packaging. The HIV-1 genome packaging is a highly

Role of NC region of Gag in recognition for packaging

It has been shown that the zinc fingers are critical in intracellular trafficking of Gag (Grigorov, Decimo et al. 2007) and in the recognition of cellular partners during budding (Popov, Popova et al. 2008;Dussupt, Javid et al. 2009). Additionnaly, mutations in NCp7 result in late RTion suggesting that NCp7 via its role in the Gag-Gag interactions can control temporally triggering of the reverse transcription (Didierlaurent, Houzet et al. 2008;Houzet, Morichaud et al. 2008).

All evidences from in vivo and in vitro studies showed that the specific interaction between viral genome and Gag is mainly displayed by its nucleocapsid domain [START_REF] Berkowitz | Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays[END_REF][START_REF] Dannull | Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues[END_REF][START_REF] Luban | Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA[END_REF][START_REF] Geigenmuller | Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence[END_REF]. Impairing zinc-binding caused an increased ratio of spliced to unspliced vRNA in virus particles and replacement of a single zinc-coordinating residue with a non-zinc coordinating one can lead to assembly of virus particles lacking vRNA [START_REF] Gorelick | Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence[END_REF]Aldovini and Young 1990;Gorelick, Nigida et al. 1990;[START_REF] Berkowitz | Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays[END_REF]Poon, Wu et al. 1996;[START_REF] Schmalzbauer | Mutations of basic amino acids of NCp7 of human immunodeficiency virus type 1 affect RNA binding in vitro[END_REF][START_REF] Damgaard | Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions[END_REF][START_REF] Cruceanu | Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins[END_REF][START_REF] Wu | Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications[END_REF]).

These results suggest that the specificity of interaction with the gRNA is provided by the zinc fingers that helps in discriminating viral genomic RNA from spliced RNA species (Aldovini and Young 1990;[START_REF] Zhang | Analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain[END_REF][START_REF] Accola | Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain[END_REF].

Gag-mediated RNA dimerization and packaging

As shown on (Figure 7), an overlap can be seen between the cis-acting elements required for RNA dimerization, that are extended downstream Gag ORF, and the transacting elements i.e.

Gag to achieve specific packaging of the viral RNA genome. Thus, the RNA elements important for packaging of HIV-1 genomic RNA, have been mapped to the highly conserved and structured 5' untranslated region and furthermore, extends into the Gag coding sequence [START_REF] Lever | Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions[END_REF]Aldovini and Young 1990;[START_REF] Clavel | A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology[END_REF][START_REF] Luban | Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein[END_REF][START_REF] Buchschacher | Human immunodeficiency virus vectors for inducible expression of foreign genes[END_REF][START_REF] Hayashi | RNA packaging signal of human immunodeficiency virus type 1[END_REF][START_REF] Luban | Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA[END_REF][START_REF] Mcbride | Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo[END_REF]. Moreover, this region that is essential for RNA packaging (SL1 to SL4) (Clever, Sassetti et al. 1995;[START_REF] Mcbride | Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo[END_REF]) also overlaps the region required for RNA dimerization. In fact, this dimerization occurs in the cytoplasm [START_REF] Moore | Dimer initiation signal of human immunodeficiency virus type 1: its role in partner selection during RNA copackaging and its effects on recombination[END_REF]) before the encapsidation process suggesting that this dimerisation process also participates to the selection of the viral RNA among the cellular RNAs [START_REF] Nikolaitchik | Dimeric RNA recognition regulates HIV-1 genome packaging[END_REF]).

3.4

Viral RNA nuclear transport and packaging

Role of nuclear trafficking of Gag in genome packaging

Unfortunately, the mechanisms related to the transport of HIV-1 gRNA throughout the cell to the assembly site at the PM are poorly understood. However, two mechanisms have been described

for cytoplasmic transport of cellular mRNAs like active transport along the cytoskeleton or passive diffusion [START_REF] Gaudin | HIV trafficking in host cells: motors wanted![END_REF]).

In the first case, RNA was shown to move along with viral Gag via endosomal trafficking pathways for reaching the sites of assembly at the PM [START_REF] Basyuk | Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles[END_REF][START_REF] Swanson | Retrovirus RNA trafficking: from chromatin to invasive genomes[END_REF][START_REF] Lehmann | Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning[END_REF]Molle, Segura-Morales et al. 2009). This hypothesis becomes more prominent with the fact that the Psi domain within the gRNA directs its targeting to a perinuclear/centrosomal site where it interacts with Gag prior to an eventual transport to the assembly site, and that its absence leads to inappropriate subcellular localization [START_REF] Poole | HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET[END_REF][START_REF] Swanson | Retrovirus RNA trafficking: from chromatin to invasive genomes[END_REF]. Additionally, some specific cellular factors were also reported to direct viral structural proteins and gRNA to the site of assembly including like the Rab family of small GTPases [START_REF] Rey | HIV-1 Gag protein associates with F-actin present in microfilaments[END_REF], Staufen (hStau), a double stranded non sequence specific RNA binding protein [START_REF] Mouland | The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation[END_REF][START_REF] Chatel-Chaix | Identification of Staufen in the human immunodeficiency virus type 1 Gag ribonucleoprotein complex and a role in generating infectious viral particles[END_REF]).

In contrast, diffusion/trapping model was proposed using total internal reflection fluorescence microscopy (TIRF) (Ivanchenko, Godinez et al. 2009;[START_REF] Jouvenet | Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles[END_REF][START_REF] Jouvenet | Visualizing HIV-1 assembly[END_REF]. Using this approach combined with fluorescently labeled and photoconvertible HIV-1 components, it was shown that a complex of few Gag molecules (<12)

and a viral RNA dimer targets the PM with subsequent irreversible anchoring, while displaying lateral mobility in the plane of the PM. This phenomenon ceases as further Gag molecules accumulate till reaching the critical number required for complete virion assembly [START_REF] Jouvenet | Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles[END_REF]). However, without Gag, the gRNA was found to be highly diffusing moving in and out near the PM. Additionally, mutations interfering with Gag multimerization and assembly, resulted in dissociation of the gRNA after being anchored at the PM. In line with this model, the group of Wei-Shau Hu et al recently shows that HIV RNA lacks directionality when moving in the cytoplasm suggesting a diffusive movement and intact cytoskeletal structure was not needed for this motion [START_REF] Chen | Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein[END_REF]).

Role of GagNC in assembly (reviewed in (de Rocquigny, El Meshri et al. 2014))

Early 90s, the NC domain of Gag was shown involved in Gag assembly and release of particles.

In fact, mutation of residues involved in zinc chelation (CCHC domain) or mutating the aromatic residues Phenylalanine16 and Tryptophan 37 of the ZFs caused a large decrease in particle production [START_REF] Gorelick | Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence[END_REF]Aldovini and Young 1990;Dorfman, Luban et al. 1993). Next, studies were carried out using the two-hybrid systems [START_REF] Franke | Specificity and sequence requirements for interactions between various retroviral Gag proteins[END_REF] and recombinant baculoviruses [START_REF] Carriere | Sequence requirements for encapsidation of deletion mutants and chimeras of human immunodeficiency virus type 1 Gag precursor into retrovirus-like particles[END_REF] it was observed that the smallest domain identified for Gag oligomer formation was encompassing the C-terminal domain (CTD)

of CA [START_REF] Wills | Form, function, and use of retroviral gag proteins[END_REF][START_REF] Strambio-De-Castillia | Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis[END_REF] and GagNC.

Next, even though other domains in the matrix [START_REF] Hong | Assembly-defective point mutants of the human immunodeficiency virus type 1 Gag precursor phenotypically expressed in recombinant baculovirus-infected cells[END_REF][START_REF] Mammano | Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis[END_REF], and the spacer peptide1 SP1 [START_REF] Krausslich | The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity[END_REF] were found essential for Gag assembly [START_REF] Gottlinger | The HIV-1 assembly machine[END_REF][START_REF] Adamson | Human immunodeficiency virus type 1 assembly, release, and maturation[END_REF], the mechanism whereby the GagNC participates in Gag assembly lies in the interaction between GagNC and nucleic acids. This interplay appears to contribute to assembly by tethering Gag molecules onto the RNA promoting Gag-Gag interaction [START_REF] Burniston | Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsiddimer interface and the basic region of matrix protein[END_REF][START_REF] Cimarelli | Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues[END_REF]Ott, Coren et al. 2009). To model homotypic GagNC interactions driven by the RNA and GagNC, GagNC was substituted for the bacillus subtilisis Apo MTRB protein that endowed protein-protein interaction and formation of particles at levels similar to the wt Gag was determined [START_REF] Antson | The structure of trp RNA-binding attenuation protein[END_REF]. This suggests the essential role of GagNC was to make protein-protein contacts.

These results were re-confirmed by the use of a Gag construct where GagNC was substituted by the leucine zipper of human CREB DNA binding domain (Gag-ZIP) [START_REF] Zhang | Analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain[END_REF][START_REF] Chang | HIV-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly[END_REF][START_REF] Wang | Transcriptional/epigenetic regulator CBP/p300 in tumorigenesis: structural and functional versatility in target recognition[END_REF] or where GagNC was replaced by the Coat protein of the MS2 RNA bacteriophage [START_REF] Dykeman | Simple rules for efficient assembly predict the layout of a packaged viral RNA[END_REF]. In all cases, viral particles can be formed by a recombinant Gag in the absence of a specific RNA partner [START_REF] O'carroll | Elements in HIV-1 Gag contributing to virus particle assembly[END_REF]. Moreover, the impact of NC on Gag-Gag interaction was tested using the ability of wt Gag to rescue non-myristylated Gag at the PM [START_REF] Morikawa | Complete inhibition of human immunodeficiency virus Gag myristoylation is necessary for inhibition of particle budding[END_REF][START_REF] Ono | Relationship between human immunodeficiency virus type 1 Gag multimerization and membrane binding[END_REF][START_REF] Li | Myristoylation is required for human immunodeficiency virus type 1 Gag-Gag multimerization in mammalian cells[END_REF][START_REF] Kawada | Dominant negative inhibition of human immunodeficiency virus particle production by the nonmyristoylated form of gag[END_REF]. Since nonmyristylated Gag proteins do not interact with membranes, different N-myristly defective mutants like ΔNC Gag and ΔCA-CTD labeled mutants were co-transfected with wt Gag. No FRET was observed with the Myr -ΔCA-CTD Gag mutant which is in agreement with the observations on homotypic complexes and thus confirming the key role of the CA-CTD domain in Gag oligomerization. Similar results Myr -ΔNC Gag mutants were obtained indicating that the NC domain is required for Gag-Gag interactions in the absence of membrane binding.

Assembly of recombinant Gag in vitro

To gain insights into the process of assembly, in vitro systems have been generated that recapitulate capsid formation outside the context of the cell by using purified recombinant Gag at high concentrations (∼1mg/ml, or 20 μM) in buffer known as HIV-1 myr(-)GagΔp6. More recently, Mckinstry et al., have managed to purify full length Gag (myr(-)Gag) and could be crucial in answering these questions [START_REF] Mckinstry | Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli[END_REF]. By using myr(-)GagΔp6 it was shown that Gag is in a monomer-dimer equilibrium in solution, while (myr(-)Gag) showed that it exist in trimeric form [START_REF] Ekram | Specific recognition of the HIV-1 genomic RNA by the Gag precursor[END_REF].

By using this in vitro approach it was established that Gag can assemble spontaneously in presence of nucleic acid, i.e. un-catalyzed and without the input of energy, still there are a number of key differences between the recombinant assembly system and the intracellular environment [START_REF] Ellis | Macromolecular crowding: an important but neglected aspect of the intracellular environment[END_REF]. Another set of studies has been performed in assembly of newly synthesized Gag using cell extracts, such as rabbit reticulocyte extract [START_REF] Sakalian | Synthesis and assembly of retrovirus Gag precursors into immature capsids in vitro[END_REF][START_REF] Spearman | Human immunodeficiency virus type 1 capsid formation in reticulocyte lysates[END_REF] or wheat germ extract [START_REF] Lingappa | A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system[END_REF][START_REF] Singh | Effect of mutations in Gag on assembly of immature human immunodeficiency virus type 1 capsids in a cell-free system[END_REF][START_REF] Zimmerman | Identification of a host protein essential for assembly of immature HIV-1 capsids[END_REF], and by linking Gag assembly to translation. It was observed that the cell-free assembly system closely recapitulates the process of assembly that occurs in cells [START_REF] Lingappa | A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system[END_REF][START_REF] Singh | Effect of mutations in Gag on assembly of immature human immunodeficiency virus type 1 capsids in a cell-free system[END_REF].

Further, studies revealed that capsid assembly in the cell-free system is energy-dependent, that ATP hydrolysis and not just ATP binding was likely required. This energy requirement is posttranslational and therefore separate from the well-understood requirement for energy during translation [START_REF] Lingappa | A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system[END_REF]. This energy-dependent in cells implies that the mechanism of assembly in cells differs from the spontaneous assembly observed with recombinant Gag in vitro and also supported by the energy-dependence of retroviral capsid assembly in the cell-free system and in cells [START_REF] Weldon | Type D retrovirus capsid assembly and release are active events requiring ATP[END_REF][START_REF] Dooher | Conservation of a stepwise, energy-sensitive pathway involving HP68 for assembly of primate lentivirus capsids in cells[END_REF], and by the identification of an ATP-binding protein, ABCE1, that facilitates assembly in cells [START_REF] Zimmerman | Identification of a host protein essential for assembly of immature HIV-1 capsids[END_REF].

Incorporation of other viral components

Other cellular and viral components important for the assembly and infectivity of virus, are

Virus particle budding and release

The terminal stage of the viral life cycle starts with budding and the assembled viral particle induce a membrane fission event to completely separate the viral envelope from the infected cell surface [START_REF] Weiss | The role of cellular factors in promoting HIV budding[END_REF]. To catalyze this egress, the virus hijacks the host ESCRT pathway [START_REF] Morita | Retrovirus budding[END_REF]Bieniasz 2009). In cell, ESCRT (endocytic sorting complex required for trafficking) factors are implicated in membrane fission reactions to release vesicles into endosomal multivesicular bodies and to separate daughter cells along the cytokinesis (Sundquist and Krausslich 2012). ESCRT factors belong from the VPS system, composed of proteins called class E, grouped into five major complexes: HRS-STAM complex, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III as well as the accessory proteins VPS4 and ALIX (Fig. 28) [START_REF] Martin-Serrano | Host factors involved in retroviral budding and release[END_REF]. As mentioned earlier, impaired viral particle release takes place when defects in HIV-1 p6 domain occur (Figure 29). With the help of its two different domains referred as "late domain" motifs, Gag p6 recruit early-acting ESCRT factors

Gag-TSG101 pathway

The primary pathway involves the late "L" domain of Gag (PTAP motif of p6), which recruits and binds directly to Tsg101 (Tumor susceptibility gene 101), a subunit of ESCRT-I, heterotetramer that also contains VPS28, VPS37 and MVB12 (Martin-Serrano and Neil 2011;

Weiss and Gottlinger 2011). Tsg101 was originally identified in neoplasia associated after its functional inactivation in murine fibroblasts [START_REF] Li | Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells[END_REF]. A variety of nuclear, microtubule, and mitotic spindle abnormalities have been observed in cell line that are deficient in TSG101 (SL6) [START_REF] Li | Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells[END_REF][START_REF] Xie | Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency[END_REF][START_REF] Zhong | Perturbation of TSG101 protein affects cell cycle progression[END_REF]. SL6 cells that are deficient in Tsg101 show defective endosomal trafficking [START_REF] Babst | Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking[END_REF].

TSG101 contains various sub domains notably an ubiquitin (Ub) conjugase (E2) -like domain that lacks the active-site Cys residue crucial to Ub conjugation (Figure 30) (Pornillos, Alam et al. 2002). The PTAP motif interacts with the amino-terminal of this domain (Garrus, von Schwedler et al. 2001;Martin-Serrano, Zang et al. 2001;VerPlank, Bouamr et al. 2001). On the other hand, the C-terminal region of Tsg101 contains a binding site for Vps28, and an upstream coiled-coil domain may bind Vps37 [START_REF] Bishop | TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes[END_REF][START_REF] Katzmann | Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I[END_REF][START_REF] Martin-Serrano | Role of ESCRT-I in retroviral budding[END_REF]. These three proteins, Vps28, Vps37 and Tsg101, form a complex called ESCRT-1 (Figure 28). Interestingly, this PTAP motif is also found in various proteins known to recruit ESCRT-I to endosomal membranes notably the human HRS [START_REF] Bouamr | The C-terminal portion of the Hrs protein interacts with Tsg101 and interferes with human immunodeficiency virus type 1 Gag particle production[END_REF]). Thus, in HIV, the PTAP domain of p6 mimics a cellular ESCRT-I recruiting motif and plays a role in virus budding.

Mutations in the PTAP motif disrupt this interaction (Demirov, Ono et al. 2002). Moreover, It has been shown by (Garrus, von Schwedler et al. 2001) by using small interfering RNAs that inhibition of TSG101 expression markedly impairs HIV-1 virus production. Similarly (Demirov, Ono et al. 2002), also demonstrated that overexpression of the N-terminal domain of TSG101

(TSG-5′) dramatically reduces virus release by blocking at the level of virus budding (Garrus, von Schwedler et al. 2001;Demirov, Ono et al. 2002). This blocking was found specific to HIV-1, as MLV release was unaffected by either TSG101 underexpression or TSG-5′ overexpression.

In addition to that, it has also been shown that p6 ubiquitination promotes the interaction of Gag with TSG101 (VerPlank, Bouamr et al. 2001;Pornillos, Alam et al. 2002;[START_REF] Freed | The HIV-TSG101 interface: recent advances in a budding field[END_REF];( reviewed in [START_REF] Freed | Viral late domains[END_REF]).

Gag-Alix pathway

A secondary late domain of p6 YPXL/LXXLF binds the ESCRT factor ALIX to initiate second budding pathway known as Alix-pathway. The YPXL late domain contributes significantly to HIV-1 replication but is less critical compare to the PTAP motif in most cell types [START_REF] Fujii | Functional role of Alix in HIV-1 replication[END_REF][START_REF] Eekels | Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors[END_REF]. Moreover, this motif YPXL has never been found in mammalian partners.

Alix interacts with the C-terminal helical regions of the CHMP4 proteins of ESCRT-III components (Martin-Serrano, Yarovoy et al. 2003;Strack, Calistri et al. 2003;von Schwedler, Stuchell et al. 2003). It is a modular protein that possesses a banana-shaped N-terminal Bro1 domain, a V-shaped middle domain, and a C-terminal PRR [START_REF] Fisher | Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding[END_REF]) domain.

Bro1 domain binds membranes and induces negative curvature through their convex surfaces [START_REF] Kim | Structural basis for endosomal targeting by the Bro1 domain[END_REF]) (Figure 31) while the V domain binds to LYPx n L-type L domains via a hydrophobic pocket on one of its arms [START_REF] Fisher | Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding[END_REF][START_REF] Lee | Structural basis for viral late-domain binding to Alix[END_REF][START_REF] Zhai | Structural and functional studies of ALIX interactions with YPX(n)L late domains of HIV-1 and EIAV[END_REF], thus linking HIV-1 Gag directly to the CHMP4/ESCRT-III membrane fission complex. Additionally, Alix has potential to bridge ESCRT-I and ESCRT-III through a possible binding between the UEV domain of TSG101 and PSAP motif of the ALIX PRR (Martin-Serrano, Yarovoy et al. 2003;Strack, Calistri et al. 2003;von Schwedler, Stuchell et al. 2003;[START_REF] Ku | ALIX is recruited temporarily into HIV-1 budding sites at the end of gag assembly[END_REF]) domain.

The LYPx n L-type L domain in HIV-1 p6 has a much lower affinity for ALIX [START_REF] Zhai | Structural and functional studies of ALIX interactions with YPX(n)L late domains of HIV-1 and EIAV[END_REF]) as compare to PTAP-TSG101, thus suggesting that HIV-1 budding depends primarily on the TSG101 binding site, and the ALIX binding site plays an auxiliary role [START_REF] Fisher | Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding[END_REF]). However, the role of the p6 ALIX binding site in particle release becomes critical in case of "minimal" HIV-1 Gag that lacks certain MA and CA NTD sequences (Strack, Calistri et al. 2003), which suggests that assembly defects can to some extent be compensated by the presence of an optimal L domain. Interestingly, it has been found that ALIX also interacts with the NC domain of Gag through its Bro1 domain (Popov, Popova et al. 2008;Dussupt, Javid et al. 2009), through conserved zinc finger motifs and on basic residues within GagNC (Popov, Popova et al. 2008;[START_REF] Sette | Identification of the HIV-1 NC binding interface in Alix Bro1 reveals a role for RNA[END_REF] as well as disruption of this interaction impaired ALIX-mediated HIV-1 budding. This also brings a notion that the viral RNA may serve as a bridging factor due to the ability of GagNC to interact with it.

Gag-NEDD4L pathway

A third type of L domain, PPXY (found in RSV, MLV, HTLV and MPMV, but not in HIV) can engage NEDD4 ubiquitin ligase. However, we cannot exclude the role of NEDD4 during HIV-1 budding from cells as there is relatively low ESCRT-I levels. Interaction between the carboxyterminal domain of GagCA and the NEDD4L can help ESCRT to assemble [START_REF] Chung | NEDD4L overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking PTAP and YPXL late domains[END_REF][START_REF] Usami | Efficient and specific rescue of human immunodeficiency virus type 1 budding defects by a Nedd4-like ubiquitin ligase[END_REF][START_REF] Weiss | Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag[END_REF]) and thus can also rescue the budding of HIV-1 L-domain mutants (Ono and Freed 2004;[START_REF] Weiss | The role of cellular factors in promoting HIV budding[END_REF].

HIV Budding

HIV-1 budding and release are essential for spreading viral infection. The virus usurps the host ESCRT pathway to terminate Gag polymerization and catalyze release of vesicles by membrane fission reactions (Sundquist and Krausslich 2012).

In essence, TSG101/ESCRT-I, ALIX and possibly NEDD4L functions by recruiting downstream ESCRT-III and VPS4 complexes, which in turn mediate membrane fission and recycling of ESCRT factor V. As illustrated in (Figure 32), entire ESCRT assembly process, in presence of ALIX, takes approximately 10 minutes and occurs in multiple stages, with a gradual and concomitant buildup of the Gag and ALIX proteins, followed by short (∼2 min) bursts of ESCRT-III and VPS4 recruitment immediately prior to virus budding (Sundquist and Krausslich 2012).This budding process is aided by CHMP4 subunits, possibly in complex with CHMP2 and other ESCRT-III proteins, to form inwardly spiraling filaments within the neck of the budding virus. These filaments may create closed "domes" that constrict the opposing membranes and promote fission (Sundquist and Krausslich 2012).

As far as virus release is concern, it seems that VPS4 plays an active role in it [START_REF] Baumgartel | Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component[END_REF][START_REF] Elia | Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission[END_REF][START_REF] Jouvenet | Visualizing HIV-1 assembly[END_REF], as ESCRT-III recruitment alone is insufficient for virus release [START_REF] Jouvenet | Visualizing HIV-1 assembly[END_REF]. It is possible that VPS4 helps to promote the formation of abovementioned ESCRT-III dome and/or removal of ESCRT-III subunits from the dome, thereby destabilizing hemi-fission intermediates and thus helps to drive fission to completion (Sundquist and Krausslich 2012).

67

Cellular partners interacting with Gag via NC domain

Several cellular partners of Gag have been identified that interact with Gag via NCp7 domain, including the actin protein [START_REF] Ott | Cytoskeletal proteins inside human immunodeficiency virus type 1 virions[END_REF], Staufen -1 [START_REF] Mouland | The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation[END_REF][START_REF] Chatel-Chaix | Identification of Staufen in the human immunodeficiency virus type 1 Gag ribonucleoprotein complex and a role in generating infectious viral particles[END_REF]) and several other proteins [START_REF] Ptak | Cataloguing the HIV type 1 human protein interaction network[END_REF]). However, the role of all the proteins, interacting with Gag, is not fully determined. Here we presented a list of known proteins, and their functions in the replication cycle, that interact with Gag via NC domain in Table 1: Table 1: List of cellular proteins which are interacts with Gag mainly via its NCp7 domain.

Interaction

The Gag structural polyprotein of HIV-1 orchestrates viral particle assembly in producer cells, in a process that requires two platforms, the genomic RNA on the one hand and a membrane with a lipid bilayer, on the other. Gag is formed composed of matrice (MA), capsid (CA), nucleocapsid (NCp7) containing two invariant CCHC zinc fingers and p6 domains which are essential for the formation of infectious viral particles. GagNC, in addition to its role as chaperone, was also shown essential in Gag assembly by virtue of its interaction with a wide range of nucleic acids thanks to numerous basic residues distributed throughout the protein. This nonspecific binding mode seems to contribute to the assembly by tethering Gag molecules onto the RNA generating a molecular crowding that in turn promotes Gag-Gag interactions.

Based on this knowledge, the aim of the thesis was to describe the role of the NC domain as part of Gag (GagNC) in Gag-Gag interaction that is essential for the assembly of the particles and also the role of Gag (NC) along the budding notably using quantitative fluorescent microscopy.

During the course of our studies we divide our aims into these categories: 293T cells: lineage constitute a derivative of human epithelial kidney cells transformed with adenovirus E1A, which also expresses the SV40 T antigen for the episomal replication of plasmids containing the SV40 origin and the promoter region. Ultra competent bacteria from New England's Biolabs (NEB 5-alpha #C2987H) were also used to amplify PCR products from cloning.

Plasmids

Methods

Transformation of competent bacteria and purification of plasmid

DNA

After slow thawing of the E. coli DH5α bacteria in ice, 50 µL of the competent bacteria were incubated with 1ng of plasmid of interest, in ice for 30min. The tube containing the mixture is then placed at 42 ° C for 45 seconds, and then immediately transferred back to ice for 10 minutes. This step is known as thermal shock (heat shock) and is a crucial step during exogenous plasmid transformed. After that, 950μl of sterile LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5g / L, pH 7 -DifcoTM LB, Lennox) were added. The bacteria are then placed in the incubator at 37 ° C for one hour. Finally, the bacteria are plated on Petri dishes containing LB agar medium (agar: 15 g / L -DifcoTM agar LB, Lennox) and the antibiotic which has the resistance gene in plasmid of interest and placed at 37 ° C overnight.

Ampicillin and kanamycin are used at final concentrations of 100μg/mL and 50μg/mL respectively.

The second day, the colonies having a thrust on the culture medium is inoculated into 3 ml of LB medium with antibiotic for 8 to 10 hours. This 3ml pre-culture is used to inoculate 150 ml of LB medium containing antibiotics and cultured at 37 ° C for 14 to 16 hours.

Purification of plasmid DNA is carried through the KIT Nucleobond Xtra Midi Plus® (Macherey-Nagel) and DNA concentrations were measured by using nanodrop.

Cell Culture: Passage of Hela cells, 293T

HeLa and 293T cells were grown in flasks of 75cm After removal of PBS, 2 to 3 mL of 1X trypsin was added. Due to the action of the enzyme cells will be separated from plastic wall of the culture flask. The flask is then placed in the incubator for 2 to 5 minutes at 37 ° C (optimum temperature action of trypsin) in a humid atmosphere with 5% CO 2 . After checking the proper action of enzyme on the optical bench microscope, 7-8 mL of DMEM, for inhibiting the action of trypsin, was added. The cells were recovered by pipetting and then placed in a 15 mL Falcon tube and then centrifuged for 5 minutes at 1100 rpm. The supernatant is then removed and the cell pellet was suspended in 10 mL of fresh DMEM solution and numbers of cells were counted by using a Neubauer cell (Figure 33):

Formule de la cellule de numération Neubauer

Les calculs avec la cellule de numération Neubauer deviennent faciles. Cellules totales comptées Concentration cellulaire = --------------------------x 10.000 Nombre de carrés 1 1x10 6 cells were then placed in a 75cm 2 flask in 20mL DMEM solution with (P/S + 10% FCS).

The cells were finally placed in incubator at 37 ° C in a humid atmosphere of 5% CO 2 .

Cell transfection

The transfection of HeLa or 293T cells is carried out by using a Polyplus jetPEI TM kit and a solution of 150 mM NaCl. Polyethyleneimine (JetPEI) is a polycationic agent which binds to DNA and allows the migration of DNA through the cell wall in a few steps. During this process, the polycation binds to the phosphate bone of negative DNA and make complexes. The complex formed is positive and is able to bind to the plasma membrane of negative polysaccharides. Once attached to the membrane, the complex is endocytosed. Moreover, JetPEI has sponge like properties which help its merger with the endosome and thereby inactivating the acidic hydrolases that could be responsible for DNA degradation. Then the polycation-DNA complex leaves the endosome, and goes to the nucleus to be transcribed.

The transfection of HeLa cells were performed in 6/12-wells plates or μ-Dish 35mm Ibidi (BioValley) (Table 5).

After, coverslip of 18 mm in diameter were deposited at the bottom of each well of 6/12-wells (except μ-Dish 35mm Ibidi, where the surface is pretreated to make it sterile and to improve cell adhesion). The slides were then sterilized by the addition of 70% ethanol for 5 minutes, then successive washing steps were performed by using solutions containing 1X PBS (1 x 2 mL) and two washes with DMEM (2 x 2 mL). The cells were seeded according to the calculated amount and are placed at 37 ° C (5% CO 2 ) for 24 hours.

For each test sample, two Eppendorf tubes are prepared according to specific methods detailed in The complexes were prepared by using volumes of plasmid DNA (X and Y) of interest and twice the volume of JetPEI® as compared to DNA.

The transfection experiment was conducted simultaneously by preparing two eppendorfs tubes at the same time. After a short incubation period of less than 5 minutes, the contents of tube 1 is added to the contents of the tube 1' (and not the reverse), vortexed for 15 seconds and further incubated for 20 minutes at room temperature. The content of tube was then deposited on seeded cells. After brief manual stirring of the well plate, the cells were returned to 37 ° C (5% CO 2 ) and allowed to incubate for the time necessary for the experiment (usually 12 to 24 hours for imaging experiments and 48 hours for co -IP). To prevent excessive cell toxicity due to the presence of JetPEI, it is important to change the medium of the culture after 5-6 hrs with fresh DMEM.

Fixation by PFA (paraformaldehyde)

In order to stabilize the structures (antigens) as close as possible to their live appearance, waterinsoluble molecules (cross-linking) were used to block the enzyme systems.

The fixer used in the laboratory is a non-coagulating fixative: paraformaldehyde (CH 2 O)n (insoluble polymer of formaldehyde from the group of aldehydes). It reacts essentially with proteins and amino acids, and allows the formation of methylene bridges which acts as waterinsoluble barrier.

Fixation of HeLa cells

Fixation of the cells is generally performed 12-24h post-transfection, in several stages. After removal of the DMEM medium, the wells containing the cells were washed 2 times with 1X PBS

(2 times 1 ml/well). Then they were fixed with 2 ml of 4% PFA for 10 min at room temperature under constant stirring and dark. Three successive washings of 5 minutes each, with 2 ml of 1X PBS, were then conducted.

Two options are possible:

-When cell were transfected with plasmids expressing fluorescent proteins, coverslip are mounted on slide using a drop of mounting medium (consisting of DAPI-Fluoromount-G and Fluoromount-G 50/50). The coverslip must be in contact with the mounting medium for at least 16 hours. Then we can proceed to microscopic observations, after gently surrounding the coverslip with nail-polish to prevent contamination and other movements during the observations.

-Either performs immunofluorescence.

Immunofluorescence of HeLa cells

Immunofluorescence allows observation of the expressed proteins (endogenous or overexpressed following transfection by a plasmid) cell by confocal microscopy.

After washes with 1X PBS previously described, the cells were permeabilized with 2 mL of 0.2% Triton (diluted in 1X PBS) for 10 minutes at room temperature under stirring and dark. The cells are then washed three times with 1X PBS with a gap of 5 each. Blocking of non-specific sites were performed by using 2% BSA (Bovine Serum Albumin) for 1 hour stirring under dark.

After washing with 1X PBS, 50 l of solution containing primary antibody diluted in 4% BSA solution (Table 2) is directly deposited on parafilm. The coverslips on which HeLa cells are adhered, is gently transferred on the drop of diluted antibody. Incubation lasts for 1 hour at room temperature in the dark. Two successive washings for slides with 1X PBS were then performed.

Coverslips were transferred again to a secondary antibody solution, diluted in 4% BSA solution (Table 3) thus, in the similar way as for the primary antibody and incubation was performed for 40 minutes in dark. Finally, the cells were washed three times with 1X PBS before their mounting with Fluoromount medium (SouthernBiotech).

Confocal Microscopy

The cellular localization of the proteins of interest coupled to fluorophores (eGFP, mCherry, Alexa Fluor and ReAsH) were visualized using a confocal microscope having a laser as a light source.

The exciting monochromatic laser beam is reflected by a dichroic mirror for reflecting only the selected wavelengths. The beam then enters the sample after being focused by a lens which, in this case, acts as a condenser. The laser delivers energy to the optical fluorochromes on impact, thus allowing them to move on an electronic layer of stronger energy. Fluorochromes naturally return to their ground state, emitting rays of lower energy. The wavelength of the emitted fluorescent light being greater than the excitation wavelength, the emitted light can pass through the dichroic mirror. Through a variable diaphragm ("pinhole") located in the focal plane of the lens of the optical tube, it is possible to select the rays emitted by a single plane of the preparation. The rays are then detected by the photomultiplier, which will convert the photonic signal to an amplified digital signal. A point by point image is reconstructed by the computer, by using received signals. Image acquisition is done by computer using the Leica software. The images obtained, once captured, are analyzed using the commercial ImageJ software.

The microscope used is a laser scanning confocal microscope Leica SPE-TC II (G objective 63X oil immersion).

FRET (Förster / fluorescence resonance energy transfer)

During my PhD, the FRET-FLIM measurements were perform to visualized Gag/Gag interactions and oligomerizations in fixed or live HeLa cells. To study Gag-Gag oligomerization by FRET-FLIM, cells were transfected by Gag proteins tagged by eGFP (donor) and mCherry or ReAsH (acceptor) (Figure 34), and we monitor the fluorescence lifetime of the donor at each pixel of the image.

FRET is a non-radiative process where the energy of a donor fluorophore (ex: eGFP) in the excited state is transferred to an adjacent acceptor fluorophore (ex mCherry or ReAsH). It is possible in the presence of an overlap between the emission spectrum of the donor and the absorption spectrum of acceptor (Figure 34) and also requires that the distance between the donor and acceptor should be less than 5-8 nm (Förster distance) with a favorable orientation.

These conditions of energy transfer between the donor and acceptor are only possible if the labeled proteins are very close and exclude random interactions (Figure 34). FRET can be carried out with various (donor, acceptor) couples, the most popular being eGFP as a donor and either mCherry or mRFP as acceptor [START_REF] Tramier | Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells[END_REF]Fritz, Didier et al. 2008

Bennet et al. 2013).

There are three major approaches for measuring FRET i) by fluorescence intensity measurements, ii) by the photobleaching of acceptor (acceptor photobleaching), and iii) by fluorescence lifetime (FLIM). FILM microscopy is the most robust approach to follow FRET in the live cells. We used this approach based on the Time-correlated single-photon counting (TCSPC) that acquire fluorescent lifetime decay of donor for each pixel of an image. In contrast to fluorescence intensity, the fluorescence lifetime is an absolute parameter that does not depend on the instrumentation or the local concentration of the fluorescent molecules [START_REF] Wachsmuth | Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy[END_REF]. The image was obtained in scanning mode microscopy. Therefore, the FRET efficiency at each pixel can be directly determined from the decrease of the fluorescence lifetime of the donor in comparison to its natural lifetime.

In practice, the donor fluorophore is excited by a pulsed laser, and its fluorescence decay is recorded by TCSPC to measure its fluorescence lifetime. The variation of the lifetime of the donor in the presence of the acceptor can calculate the efficiency of energy transfer through the equation:

Where τ (d) is the lifetime of the donor alone and τ (ad) is the lifetime of the donor in the presence of the acceptor.

The imaging fluorescence lifetime measurements are performed over a home-made scanning microscope using a two-photon excitation. This setup, developed in the laboratory ( As shown in the above image, we observe two lifetime values in our experimental conditions.

The lower lifetime values at the plasma membrane (yellow color) compared to the cytoplasm suggests (green to blue color) that either the eGFP and mCherry tags are closer to each other in the oligomers (giving a higher FRET efficiency) or that a higher fraction of Gag proteins are in oligomeric form at the membrane (Figure 35). The time-resolved decays of Gag-eGFP ± Gag-mCherry were analyzed by a two component model function.

Where, τ1 and τ2 are short-lived and long-lived lifetime, respectively. Additionally, α1 and α2 are corresponding populations of short and long-lived lifetime species, respectively.

Protein Assay

The Bradford assay is a spectroscopic assay to measure the concentration of protein in solution using colorimetry. This assay is based on the fact that Coomassie blue (G250) absorbance change when it interacts with the basic amino acids (arginine in particular) or aromatic proteins.

Bradford solution is obtained by mixing 1 ml of the "BioRad Protein Assay" solution with 4mL of milliQ water. 200 µL this Coomassie blue solution is then deposited into the wells of a 96 well plate (200 µL / well). To establish standard curve, a solution of BSA (Bovine Serum Albumin)

was diluted to one-tenth in Tris buffer and an increasing amount of BSA is then placed in the wells of a 96 well plate (Figure 36). Similarly, solutions containing proteins (cell lysate) are also diluted to 1/20 in Tris and 10 µl of this protein solution are then used (Figure 36).

Co-immunoprecipitation

Three wells of a 6-well plate were transfected with the same sample. 48 hours post-transfection the DMEM medium is removed from the wells by a water pump. Then, the cells are rinsed with 1 mL of 1X PBS, trypsinized by adding 1 ml of trypsin (1X) and incubated at 37 ° C for 2 to 3 minutes. Then, 2 ml of DMEM medium is added and the total volume of the three wells, corresponding to the sinlge sample, is transferred into one 15 ml Falcon tube. The recovered cells were centrifuged at 14,000 rpm for 3 min. Then, the supernatant is aspirated and the pellet is lysed by the addition of 1mL of sample lysis buffer (10mM Tris HCl, 150mM NaCl, 1mM

EDTA, 1% NP40, 0.05% SDS) containing protease inhibitors (Complete Mini, EDTA-free Protease Inhibitor Cocktail Tablets, Roche) for 30 minutes at 4°C to be then transferred to

Eppendorf tubes and centrifuged for 10 minutes at 4°C at 14,000 rpm. The supernatant is recovered and the amount of total protein was assayed by the Bradford method. The supernatant is divided into two parts (as Figure 37): 20 micrograms of protein are used in 'input' and 1 mg of total proteins is used for immunoprecipitation (IP).

'The input' which shows that the protein of interest is present in the lysate before immunoprecipitation was also checked. The proteins of the 'input' are directly denatured for 5 min at 95 ° C in a solution of 1X Laemmli (Tris-HCl 250 mM, 100% glycerol, 20% SDS, 5 mg of bromophenol blue) containing 100 mM DTT.

For the IP, proteins were incubated with 1 µg of the antibody of interest (anti-Gag p24, anti-HA or anti-TSG101) and stirred for 2 hours on a wheel at 4 ° C. Meanwhile, protein A coated (Dynabeads) magnetic beads (by 50µL for each sample) were washed in 1X PBS (700μl) and then equilibrated by two successive washes in NP40 buffer (described above). To separate the beads from washing solutions, the tubes are placed on a magnetic rack. The prepared beads are incubated with the IP tubes and placed on the wheel under stirring at 4 ° C for one hour. Using the magnetic rack,beads are then washed three times with 500 µL of NP40 lysis buffer, taken up in a solution composed of 50 µL of Laemmli and DTT (32.5 µL H2O,5μL DTT,12.5μL 4X Laemmli) and denatured for 5 minutes at 95 ° C. Storage takes place at -20 ° C prior to analysis by Western blot (Figure 37). 2A). Then the membrane was washed 3 time for 10 minutes with 10 mL of TBS-T and incubated for one hour at room temperature with a secondary antibody coupled to HRP (horseradish peroxidase) (Table 2B). For exposure, two 50 mL Falcon tubes 94 were prepared, tube A containing 17 µL of coumaric acid (90 mM), 40 µL of luminol (250 mM), 400 µL of 1 M Tris, pH 8.5 and 3.5 ml of water and tube B containing 2.5µL hydrogen peroxide, 400 µL of 1 M Tris, pH 8.5 and 3.6 mL of water. These two tubes were mixed and the solution is deposited on the membrane for one minute and then removed. The membrane is placed in a cassette and the exposure is made in the darkroom or using camera-las4000 GE Healthcare without requiring the cassette. The peroxidase catalyzes the oxidation of luminol in the presence of H 2 O 2 . Luminol returned to its reduced state by emitting photon (chemiluminescence) that were analyzed in LAS4000. The membrane can be reused for other proteins detected after three washes of 10 min each with TBS-T, it is dehybridized with 10 mL of a solution 'restore most western blot stripping buffer' (ThermoScientific) for 15 min at room temperature under constant stirring. The membrane is then washed 3×10 min in TBS-T and placed under agitation. Then, the steps of blocking and incubation with the solutions containing the primary and secondary antibodies as described above are restarted.

Mutagenesis

To perform all mutants of Gag, we design primer as mentioned before. For that, I used the protocol of "Site-Directed Mutagenesis Kit" which consists of three steps:

-The first step is a PCR reaction (see Table 5) for the amplification of the plasmid to be mutated with their phosphorylated primers whose design is as shown in (Figure 38). Amplification is carried out using a thermocycler with the undermentioned program (Figure 39).

The sequences of the primers used, are given in Table 4. -Single step of annealing the PCR product with T4 DNA ligase.

-Circularized of transformation DNA in ultra-competent bacteria (NEB), then verification of the mutation by sequencing of the plasmid. The undermentioned solutions were prepared and the pointwise protocol was followed:

ReAsH/FlAsH Staining

-OPTI-MEM+GlutaMAX 1x (invitrogene): Store the solution at 4°C. Use it at RT for labelling.

-Flash 1.25µM: stock Flash 800x (1x =1.25µM), dilute 1.25µl of pure Flash in 2ml of OPTI-MEM+GlutaMAX 1x (depends on the volume).

-BAL wash Buffer (2, 3-mercaptopropanol): dilute 64µl of BAL pure in 6.4ml of OPTI-MEM+GlutaMAX 1x should be handled under the hood.

Protocol for Labelling

Before labelling media was removed and undermentioned steps were performed:

-Rince 2 times by 2 ml of OPTI-MEM+GlutaMAX 1x.

-Remove OPTI-MEM+GlutaMAX 1x, add 500µl of ReAsH/FlAsH.

-Incubate 1h at 37°C.

-Rince with 2 ml of OPTI-MEM+GlutaMAX 1x.

-Incubate 10min with 1ml of BAL wash buffer (BAL/ OPTI-MEM+GlutaMAX 1x) at RT in dark atmosphere.

-Rince with OPTI-MEM+GlutaMAX 1x.

For non-fixed cells, microscopic observation could be performed immediately or after several hours. In the later case, the OPTI-MEM+GlutaMAX were replaced by cell culture medium (DMEM,) and cells were placed again at 37°C. At the time of observation, DMEM was again replaced by OPTI-MEM+GlutaMAX 1x and observations were made.

103

The first aim of this work was to analyse the influence of the NC zinc finger (ZF) on the intracellular localization and assembly of HIV-1 Gag. Therefore, we used fluorescent Gag derivatives such as Gag-eGFP and Gag-mcherry, where the fluorophore was inserted next to the MA domain. The monitoring of protein-protein interaction in cellular context can be performed by different techniques such as by fluorescence spectroscopy that needs to extract proteins, by intensity after photobleaching or radiometric images but with many controls (cross talk, filters, level of expression….). All these approaches were used to monitor Gag-Gag interaction and a review was published to compare all this techniques (publication 1). Nevertheless, to get rid of these drawbacks, during my thesis, Gag assembly was directly visualized by using the FLIM approach combined with FRET. This technique measures the fluorescence decay of the donor at each pixel of the image and from the decay function, an average fluorescence lifetime is derived.

Moreover, using a multi-exponential decay analysis, the fluorescent lifetime and the amplitude are obtained to determine the presence of interacting and non interacting population of proteins.

Using this approach, detailed analyses of FRET images revealed a homogenous distribution of In this article, we monitored the impact of NCp7 domain on the trafficking of Tumor susceptibility gene 101 (TSG101). We first validated that the removal of p6 domain of Gag results in a dramatic decrease of Gag-TSG101 interaction by immune-precipitation and FRET-FLIM. By using similar techniques, we found that the NCp7 alone or as a domain of Gag (GagNC) was required for Gag to interact with TSG101 suggesting an interplay between NC and L domain of Gag in the process of viral budding and release. The NCp7 mediated interaction between Gag and TSG101 necessitates at least one of the two zinc fingers since removal of a singular zinc finger within GagNC or within NCp7 does not impact the interaction with TSG101.

Next, by analyzing amplitudes associated to the short-lived lifetime in the cytoplasm, in vesicles and at the PM, it was concluded that Gag is the main driving force for TSG101 trafficking.

Moreover, the removal of RNA does not abrogate Gag-TSG101 interaction and thus, this NC-TSG101 interaction seems highly contrasted to the interaction of NC and Bro 1 occurring in the LYPXnL pathway. This leaves to imply that the two mechanisms maybe not redundant but rather that the NC could recruit simultaneously both TSG101 and ALIX proteins in order to best optimize the chances of the virus to bud. This brings to a hypothesis which implies that the two mechanisms, Gag/TSG101 and Gag/ALIX, maybe not redundant but concordant, in a way that the NC could recruit simultaneously both TSG101 and ALIX proteins in order to best optimize the chances of the virus to bud.

In summary, we have used a series of biochemical assays to underline the role of NCp7 domain of Gag for the recruitment of TSG101. These approaches have provided evidences that the complex formation of Gag-TSG101 get strengthened by direct interaction of TSG101 with either free NC or NC as GagNC and by the virtue of GagNC, Gag trafficking is responsible for the docking of TSG101 to the PM.

Publication 3:

Influence of the nucleocapsid region of the HIV-1 Gag polyprotein precursor on the trafficking of the Tumor susceptibility gene 101.

Introduction

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is composed of four domains, that are the matrix (MA), capsid (CA), nucleocapsid (NC) flanked by SP1 and SP2 and p6, all involved in viral particle assembly and budding (Sundquist and Krausslich 2012). This complex process takes place soon after Gag synthesis where the MA region targets Gag oligomers to the plasma membrane (PM) through the interaction of its N terminal myristate and several basic residues located close to the N terminus. The CA region notably its C-terminal domain (CTD) together with SP1 is the major player in Gag oligomerization, further facilitated by the basic NC region interacting with the genomic RNA and considered to be the initial assembly plateform (Muriaux and Darlix 2010). At least, the C terminus of Gag corresponding to p6 is required for the incorporation of the accessory protein Vpr. This protein also contains two short sequences PTAP (Gottlinger et al. 1991;[START_REF] Huang | p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease[END_REF] and LYPX n L [START_REF] Puffer | Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein[END_REF]Strack et al. 2003) referred to as the 'late assembly domains' which binds TSG101 and ALIX (Garrus et al. 2001;Martin-Serrano et al. 2001;VerPlank et al. 2001;Strack, Calistri et al. 2003;von Schwedler et al. 2003). These sorting proteins which belong to the cellular ESCRT machinery are transiently recruited at the HIV-1 budding site to facilitate membrane fission and viral particle egress [START_REF] Bleck | Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding[END_REF][START_REF] Prescher | Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites[END_REF]. In that respect, the Tsg101/Gag-PTAP interaction is considered to be the major determinant for HIV-1 particle release [START_REF] Bieniasz | Late budding domains and host proteins in enveloped virus release[END_REF]Sundquist and Krausslich 2012). Tsg101 is a member of the ESCRT-1 complex that initiates the sorting of surface proteins into multivesicular bodies (MVB) with the help of ESCRT-III and the activity of VPS4 AAA-type ATPase. Either depletion or over-expression of TSG101 blocked HIV-1 budding and particle release (Garrus, von Schwedler et al. 2001;Demirov et al. 2002;[START_REF] Goila-Gaur | Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression[END_REF]). Moreover, it has been shown that the interaction of TSG101 and the PTAP motif is mediated by the N-terminal ubiquitin (Ub) conjugase ( E2) -(UEV) like domain of TSG101 (Pornillos et al. 2002).

As far as the role of PTAP is concerned, recent studies suggested that this motif facilitates virus budding and release in a context-dependent manner where an optimal activity requires its positioning near GagNC. This implies that it cooperates with other Gag regions to achieve an efficient viral release [START_REF] Yuan | Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses[END_REF][START_REF] Strack | Late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis[END_REF]Martin-Serrano et al. 2003). In line with this, addition of the NCp7-p1 (NCp9) region to the Gagp6-Leucine zipper (Gag Z) was found to turn it into a transdominant negative inhibitor of the ESCRT pathway. This clearly shows that NCp9 should play an important role in the ESCRT-dependent HIV-1 particle release [START_REF] Popova | Human immunodeficiency virus type 1 nucleocapsid p1 confers ESCRT pathway dependence[END_REF]. In support of this, mutating the basic residues of NC results in an inhibition of Gag particles release (Cimarelli et al. 2000;[START_REF] Ott | Elimination of protease activity restores efficient virion production to a human immunodeficiency virus type 1 nucleocapsid deletion mutant[END_REF]Ott et al. 2005 ), confirming that the NC region could cooperate with p6 as late assembly domain in the PTAP/TSG101 sorting pathway [START_REF] Dussupt | Basic residues in the nucleocapsid domain of Gag are critical for late events of HIV-1 budding[END_REF][START_REF] Bello | Budding of retroviruses utilizing divergent L domains requires nucleocapsid[END_REF]. In agreement with this notion, in the pNL4.3 context, Gag expressing the first 433 amino acid (MA, CA, p1 and NC) and so lacking the p6 domain was shown to bud normally [START_REF] Gan | HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol[END_REF].

Recently Mougel and co-workers reported that GagNC and TSG101 were in close proximity and showed the importance of the second zinc finger of NC in this proximity. Nevertheless, the low resolution of the Duolink approach (<40nm) prompt us to determine if this proximity could correspond to an interaction between GagNC and TSG101. Therefore, co-immunoprecipitation and FLIM-FRET experiments were performed on cells expressing TSG101 and a series of Gag constructs with deletion in NC and p6 regions. Results showed that both NC zinc fingers are important for the Gag-TSG101 interaction. However, deleting the NC zinc fingers did not have an impact as drastic as deleting the entire p6 region indicating that the NC domain of Gag provides a supportive role for the interaction between p6 and TSG101. Of note, the cellular localization of the Gag-TSG101 relies on Gag trafficking and thus not restricted to the PM.

Interestingly, Gag trafficking is dependent on the NC domain, and thus localization of the TSG101/Gag complex is also dependent on NC. Collectively, our results support a model where NC cooperates with p6 to recruit TSG101, thus giving an insight into the functional link between the p6 late domain motifs and NC as well as the regulatory role of NC domain in the course of recruiting components of the cellular machinery necessary for HIV-1 particle budding and release.

Materials and Methods

Plasmid DNA. The human-codon-optimized Pr55 Gag-TC encoding plasmid was kindly provided by David E. Ott (National Cancer Institute at Frederick, Maryland) (Rudner et al. 2005). Gag ∆ZF1, Gag∆ZF2, Gag∆ZF1∆ZF2, Gag∆NC and Gagp6 (Fig. 1) deletion mutants were constructed by PCR based site-directed mutagenesis using Gag-TC as substrate (El Meshri et al. 2015). pNL4-3 expressing wild type Gag and as well as zinc fingers deleted Gag were already published (Grigorov et al. 2007). HA-Tsg101 and eGFP-Tsg101 (Fig. 1) were purchased from NIH AIDS Reagent Program. The integrity of all plasmid constructs was verified by DNA sequencing.

Cell culture and plasmid DNA transfection. HeLa cells (from ATCC, ref CCL-2 Amp, HeLa;

Cervical Adenocarcinoma; Human) were cultured in Dulbecco's modified eagle medium supplemented with 10% fetal calf serum (Invitrogen Corporation, Cergy Pontoise, France) and 1% of an antibiotic mixture (penicillin/streptomycin: Invitrogen Corporation Pontoise, France) at 37°C in a 5% CO2 atmosphere. HeLa cells were transfected or co-transfected using jetPEITM (Life Technologies, Saint Aubin, France) according to the supplier's recommendations. To keep a constant amount of transfected DNA, each transfection assay was supplemented with pcDNA3 (Invitrogen Corporation, Cergy Pontoise, France).

Immunoprecipitation and western blotting. HeLa cells were seeded (6 10 5 cells) and transfected with 1µg of plasmid encoding HA-TSG alone or with 1µg of plasmid encoding either Gag-TC, Gag∆ZF1-TC, Gag∆ZF2-TC, Gag∆ZF1∆ZF2-TC, Gag∆NC-TC, Gagp6-TC or NCp7-eGFP (Fig. 1) using jetPEI TM (Life Technologies, Saint Aubin, France) according to the supplier's recommendations. 48 hours post-transfection, cell were trypsinized and resuspended in lysis buffer (10mM Tris HCl, 150mM NaCl, 1mM EDTA, 1% NP40, 0.05% SDS) containing protease inhibitors (Complete Mini , EDTA-free Protease Inhibitor Cocktail Tablets, Roche) for 30 minutes at 4° C. After centrifugation at 14,000 rpm, the amount of protein was determined by the Bradford method and 20 µg of protein were used for input and 1 mg incubated with rabbit anti-HA (Abcam ab9110) for 2 hours at 4°C under continuous agitation. Meanwhile, 50µL protein A coated (Dynabeads) magnetic beads were washed in 1X PBS and incubated with samples for 1.5µg of plasmid encoding for wild-type Gag-TC or its cognate mutants Gag∆ZF1-TC, Gag∆ZF2-TC, Gag∆ZF1∆ZF2-TC, Gag∆NC-TC, Gagp6-TC, NC-TC. Biarsenical labeling was achieved 24 h post-transfection as previously reported (Fritz et al. 2010). Briefly, a biarsenical solution was prepared by diluting 1.25µl of pure ReAsH 2 mM in 2ml of OPTI-MEM. The ReAsH solution was applied to each coverslip, incubated for 1 h at 37°C followed three washes with OPTI-MEM. Then, cells were incubated 10min. at 37°C with BAL buffer (2, 3mercaptopropanol) and washed by OPTI-MEM. Live cells were after fixation by using 4% paraformaldehyde (PFA).

Confocal Microscopy. HeLa cells were transfected in 6-well plate containing a 18 mm cover slips with a mixture containing 0.25µg of plasmid eGFP-TSG, 0.5µg of plasmid expressing Gag (or mutated Gag) or pNL43 wild-type or mutated in Gag, as described in (Grigorov, Decimo et al. 2007), and 0.25µg of plasmid expressing Gag-mCH (or the corresponding mutated Gag-mCherry). At 24 h. post-transfection, the cells were extensively washed, fixed with a 4%

PFA/PBS solution and kept in PBS at 4°C until observation with a Leica SPE equipped with a Leica 63x 1.4NA oil immersion objective (HXC PL APO 63x/1.40 OIL CS). The eGFP images were obtained by scanning the cells with a 488 nm laser line and a 500 to 555 nm band-pass for emission. For the mCherry images, a 561 nm laser line was used with a 570 to 625 nm bandpass. The same procedure was applied for HA-TSG but using rabbit anti-HA (Abcam ab9110)

followed by anti rabbit conjugated with Alexa 488 (Invitrogen Corporation, Cergy Pontoise, France, ref : A11008).

Fluorescence Lifetime Imaging Microscopy (FLIM).

HeLa cells were transfected in µ-Dish IBIDI (Biovalley, France) with a mixture containing 0.25µg of plasmid eGFP-TSG and 0.75µg of plasmid expressing Gag-TC (or mutated Gag-TC) and fixed with 4% PFA 24h. post transfection. The experimental set-up for FLIM measurements has already been described (Clamme et al. 2003;Fritz et al. 2008;[START_REF] Richert | Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy[END_REF]. Briefly, time-correlated single-photon counting FLIM measurements were performed on a home-made two-photon excitation scanning microscope based on an Olympus IX70 inverted microscope with an Olympus 60× 1.2NA water immersion objective operating in the descanned fluorescence collection mode. Two-photon excitation at 900 nm was provided by a mode-locked titanium-saphire laser (Tsunami, Spectra Physics). Photons were collected using a set of two filters: a short pass filter with a cut-off wavelength of 680 nm (F75-680, AHF, Germany), and a band-pass filter of 520 ± 17 nm (F37-520, AHF, Germany). The fluorescence was directed to a fiber coupled APD (SPCM-AQR-14-FC, Perkin Elmer), which was connected to a time-correlated single photon counting (TCSPC) module (SPC830, Becker & Hickl, Germany).

Typically, the samples were continuously scanned for about 60 s to achieve the appropriate photon statistics in order to investigate the fluorescence decays. Data were analyzed using a commercial software package (SPCImage V2.8, Becker & Hickl, Germany).

For Fluorescence Resonance Energy Transfer (FRET) experiments with eGFP, the FRET efficiency (E) was calculated according to the equation:

6 0 ) / ( 1 1 1 R R D DA E       (1)

149

where τ DA is the lifetime of the donor in the presence of the acceptor and τ D is the lifetime of the donor in the absence of the acceptor.

Results

Contribution of NC to Gag-TSG101 complex formation and trafficking to the plasma membrane

Expression of Gag NC deletion mutant (GagNC), either in the pNL4.3 backbone or from a plasmid expressing Gag, has been shown to result in a drastic reduction in the quantity of viral particles in the supernatant (Grigorov, Decimo et al. 2007;Hogue et al. 2009;Ott et al. 2009;[START_REF] Dussupt | Basic residues in the nucleocapsid domain of Gag are critical for late events of HIV-1 budding[END_REF]El Meshri et al. 2015), despite the fact that GagNC contains PTAP motif. Meanwhile, we have recently shown that GagNC can form large intracellular aggregates and oligomerize with lesser degree of compactness as compared to Gag (El Meshri, Dujardin et al. 2015). This favored the notion that deleting NC has a negative impact on the overall structure of the Gag protein and, as a consequence, potentially masking the presence of the p6 PTAP motif. To test this hypothesis, we initially followed the co-localization of wild Gag and TSG101.

Therefore, 293T cells were transfected with plasmids expressing eGFP-TSG101 alone (images ac) or with pNL4.3 (images d-f) encoding for all the viral proteins, except the ENV glycoprotein.

The anti-Gag anti body being poorly efficient for detecting Gag protein at the PM, an additional plasmid was added to pNL4.3 to produce Gag-mCherry (ratio 0.9/0.1). The fluorescent protein was inserted upstream of CA (Muller et al. 2004;Hubner et al. 2007) since insertion at this location as well as its transfection together with plasmids expressing non labeled Gag proteins was shown to moderately affect the virus morphology, Gag localization and Gag budding (Muller, Daecke et al. 2004;Larson et al. 2005;[START_REF] Lampe | Double-labelled HIV-1 particles for study of virus-cell interaction[END_REF]Jouvenet et al. 2008;Ivanchenko et al. 2009). As expected, in presence of Gag, a significant Gag-mCherry staining was monitored at the PM (image e). Since Gag-mCherry alone weakly accumulates to the PM (data not shown), this staining shows that Gag-mCherry gets embedded in Gag oligomers along the assembly process. In contrast, eGFP-TSG 101solely appears into cytosolic fluorescent dots (image a) while in the presence of Gag during the assembly process, the cytosolic fluorescence dots of eGFP-TSG101 disappeared and concentrated at the cell periphery (image d), rendering co-localization of Gag/Gag-mCherry and eGFP-TSG101 at the PM (image f). Next, we examined the localization of eGFP-TSG101 in presence of GagNC (images g-i). At 24h post transfection, aggregates of GagNC/GagNC-mCherry were observed mainly in the cytoplasm (image h). Interestingly, a partial co-localization of eGFP-TSG101 was also observed with GagNC/GagNC-mCherry (image j). Taken together, these results show that upon addition of Gag, TSG101 is relocated from the cytosol to the cell PM, and that a deletion of the NC domain of Gag prevents this relocation.

Both NC zinc fingers cooperate with p6 to promote TSG101-p6 interaction

To further characterize the role of GagNC in TSG101 recruitment, we performed immunoprecipitation experiments on cell extracts upon expression of HA-TSG101 (Fig. 3 well

2) and wild type Gag (Fig. 3, wells 3). Similar experiments were performed with GagNCp7 (well 6) and Gag containing a stop codon upstream the p6 domain (well 7). In agreement with the homogeneous distribution of GADPH (Fig 3A ), similar levels of HA-TSG101 were found in cells expressing either the protein alone (well 2), or in presence of Gag (wells 3) or of GagNCp7 (well 6) or of Gagp6 (well 7). Cell lysates were incubated with rabbit anti-HA antibodies and the transfer membrane was probed with a mouse anti-HA anti body showing similar levels of HA-TSG101 (Fig 3B,wells 2,[START_REF] Salah | Delphine Muriaux and Hugues de Rocquigny Influence of the nucleocapsid region of the HIV-1 Gag polyprotein precursor on the recruitment and trafficking of the Tumor susceptibility gene 101[END_REF][START_REF] Summers | Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1[END_REF][START_REF] Demene | 1H NMR structure and biological studies of the His23-->Cys mutant nucleocapsid protein of HIV-1 indicate that the conformation of the first zinc finger is critical for virus infectivity[END_REF]. As a control, in absence of anti-HA, no signal of Gag was observed (well 1').

The same membrane was re-probed with a monoclonal anti-Gag antibody and results revealed that Gag was efficiently captured by HA-TSG101 (well 3). In sharp contrast, GagNC was poorly precipitated by TSG101 (well 6). Interestingly, the deletion of the p6 region was found to be more drastic since only a faint reproducible signal of Gagp6 was detected in well 7. Intensity of each signal from membranes B and m C was determined and independently normalized to the signal obtained for Gag. The ratio of IP to CoIP reported in the histogram of figure 1D shows that Gagp6 and GagNC are six and two times, respectively, less efficiently iped by TSG101 as compared to wt Gag.

Next, to map the domain of NC involved in TSG101 recognition, we tested GagZF1 and GagZF2 mutants in which either the proximal or the distal NC zinc finger was deleted, (well 4 for GagZF1 and well 5 for GagZF2). To determine the role of the basic residues flanking the CCHC motifs both zinc fingers were deleted (well 8 for GagZF1ZF2). As seen on Fig 3C,

GagZF1 (well 4) and GagZF2 (well 5) were equally precipitated as compared to the wild type Gag (well 3). In sharp contrast, GagZF1ZF2 was poorly precipitated by TSG101 (well 8), the efficiency of the precipitation being similar to that obtained for GagNC (figure 1D).

Taken together, the main interaction between Gag and TSG101 is driven by p6, but also by NC:

NC contributes strongly into the recognition of TSG101 by p6.

Figure 4 Then, we examined if NC outside the context of Gag could interact with TSG101. As shown in figure 4 with the membrane blotted with anti-eGFP, HA-TSG101 was found to be able to precipitate NCp7-eGFP (well 2). Interestingly, NCp7ZF1 (well 3) and NCp7ZF2 (well 4)

were also co iped while deletion of both zinc fingers in NCp7ZF1ZF2 was detrimental for the TSG-NCp7 interaction (well 5).

Next, we tested whether the NC mediated interaction between Gag and TSG101 was RNA dependent as NC has a high affinity for nucleic acids (Darlix et al. 2014). Therefore, an RNAse treatment was applied prior to the immunoprecipitation of the sample 3 containing Gag and TSG101. As shown in figure 3C, well 2', Gag was precipitated by TSG101 in the presence of RNase, thus showing that the interaction is most probably RNA independent. All together, these results showed that both zinc fingers of NCp7 are required for the interaction between NC and TSG101.

Influence of GagNC on TSG101 cellular localization

In the previous section, the effect of Gag on TSG101 localization was monitored by immunofluorescence microscopy using anti-Gag and either anti-HA-TSG101 or eGFP-TSG101 (Martin-Serrano, Zang et al. 2001;Derdowski et al. 2004;[START_REF] Johnson | The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique nonendosomal structures[END_REF]). In the absence of Gag, TSG101 was found in the cytoplasm and accumulating in dots. Expression of TSG101 together with Gag resulted in a total accumulation of both Gag and TSG101 at the cell periphery, in agreement with the role of TSG101 during Gag budding [START_REF] Prescher | Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites[END_REF].

Figure 5 To investigate the role of GagNC in TSG localization, a plasmid expressing TSG101 was cotransfected with plasmids expressing either Gag or Gag mutants with deletions in the NC or the p6 region (Fig 1). Moreover, to monitor Gag-TSG101 interaction by means of FRET-FLIM, eGFP was used as the donor while ReAsH was the acceptor. To this end, HA-TSG101 was substituted for eGFP-TSG101 since location of eGFP at the N terminus of TGS101 does not modify its staining pattern (Fig S1). Concerning Gag, the small tetracysteine (TC) motif was added at the C-terminus of Gag to use the biarsenical-tetracysteine labeling approach [START_REF] Adams | New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications[END_REF]. In this approach, TC interacts tightly with ReAsH and both the size and the location of TC has been shown to be suitable for imaging Gag trafficking in cell (Rudner, Nydegger et al. 2005;Gousset et al. 2008;Fritz, Dujardin et al. 2010).

First, these constructs were expressed and their cellular localization was assessed by confocal microscopy. Therefore, to avoid feeble detection of Gag at the PM by anti-Gag antibody, a plasmid expressing Gag-TC or Gag-TC mutants was co-transfected with plasmid expressing Gag-mCherry and Gag-mCherry counterpart. As shown in figure 5, image a, TSG101 displays a punctuate and a faint diffused staining in the cytoplasm while in presence of Gag/Gag-mCherry, TSG101 and Gag co-localize at the PM (image b). For the GagZF1 (image c) and GagZF2

(image d) mutants, we observed a comparable co-staining of Gag and TSG101 at the PM. In addition, dots containing both partners were also detected within the cytoplasm, notably for GagZF2. Next, we examined the localization of the GagZF1ZF2 (image e) and GagNC (image f) with TSG101. An increase in diffusion in the cytoplasm, accumulation in dots or in large aggregates and only a slight co-localization at the PM were observed for co-transfected TSG101 and Gag mutants. In addition, all these staining patterns turned out to be very similar to those for Gag and GagNC mutants alone (Grigorov, Decimo et al. 2007;Hogue, Hoppe et al. 2009;El Meshri, Dujardin et al. 2015), suggesting that the localization of TSG101 at the PM essentially depends on the localization of Gag at the PM with the integrity of the NC domain and especially both zinc fingers.

Next, the cells co-expressing eGFP-TSG101 and Gagp6 were observed (image g). As shown in images g, Gag was found accumulating at the plasma membrane while TSG101 was characterized by a punctuated staining in the cytoplasm that resembles to the staining observed in cell expressing TSG101 alone (image a). Last, eGFP-TSG101 was co-expressed with NC/NC-mCherry (image g) and NCp7 was found all over the cell with partial accumulation in the nucleolar compartment, while TSG101 was observed in form of punctuated dots. These results are in agreement with the results obtained for cells expressing NCp7 (Anton et al. 2015) [START_REF] Lochmann | NC-mediated nucleolar localization of retroviral gag proteins[END_REF] or TSG101 alone (Martin-Serrano, Zang et al. 2001;Derdowski, Ding et al. 2004;[START_REF] Johnson | The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique nonendosomal structures[END_REF].

Monitoring Gag-TSG101 interaction by FLIM

In order to image Gag-TSG101 interaction and to delineate the role of GagNC in Gag-TSG101 interaction, cells co-expressing eGFP-TSG101 and Gag-TC were treated with the membranepermeable biarsenical dye ReAsH and analysed by confocal microscopy (figure S2), to check their co-localisation. In parallel, FRET is monitored by FLIM, using eGFP-TSG101 as the donor and Gag-TC-ReAsH as the acceptor. FRET between eGFP and ReAsH only occurs when they are less than 5.6 nm apart, a distance corresponding to intermolecular protein-protein interactions [START_REF] Sun | Forster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells[END_REF]. Due to FRET between eGFP and ReAsH, a decrease of eGFP fluorescence lifetime () is observed, which was determined by measuring the fluorescence decay at each pixel of the cell. Then FLIM images are built, by using false-color lifetime encoded pixels that directly described the spatial distribution of protein-protein interactions. We then monitored the viral determinant involved in Gag-TSG101 interaction in cells. Next, cells expressing Gag-TC and eGFP-TSG101 were imaged by FLIM. Analysis of the fluorescence decays using a mono-exponential model gave rise to a bimodal distribution of the lifetimes with peaks centered at 1.5 ±0.1 ns and 2.3 ± 0.2 ns and a  factor above 3. As already described [START_REF] Richert | Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy[END_REF], to obtain a better fit fluorescence decays were analyzed with a two components model: Gag (panel i), GagZF1 (panel j) or GagZF2 (panel k) showed a comparative yellow color located at the PM, the cytoplasm and in internal dots (white arrows). Concerning, GagZF1ZF2 (panel l) and GagNC (panel m), amplitudes were significantly different and dropped to 31% and 41% respectively. Interestingly, the drop in distribution of the amplitude was not homogenous and was compensated by an increase at the periphery of the PM (Figure 6, panels l and m, note the evolution from bleu to orange from the cytoplasm to the PM).

Discussion

In HIV-1, there are two different sequences located in the p6 region of Gag, namely P(T/S)AP and LYPXnL, that recruit TSG101 and ALIX, respectively, and as such are essential elements for viral particle budding [START_REF] Votteler | Virus budding and the ESCRT pathway[END_REF]. In addition to p6, when either GagNC is mutated or not in the vicinity of the PTAP sequence, virus budding was largely prevented indicating that GagNC has a role during the PTAP-mediated virus budding. In the present study, we showed that the removal of p6 resulted in a dramatic decrease of Gag-TSG101 interaction, in agreement with the seminal work showing the involvement of PTAP motif in the recruitment of ESCRT-I proteins (Garrus, von Schwedler et al. 2001;Martin-Serrano, Zang et al. 2001;VerPlank, Bouamr et al. 2001;Demirov et al. 2002;Pornillos, Alam et al. 2002).

Nevertheless, we showed a significant level of Gagp6 iped by TSG101 highlighting that another region of Gag is probably involved in the interaction. Our results suggest that NCp7 alone or as GagNC, is this region possibly engaged in interplay with the L domain required for virus budding and release.

This NC mediated interaction between Gag and TSG101 is dependent on the presence of at least one of the two zinc fingers, since removal of one zinc finger in GagNC or NCp7 does not alter the interaction with TSG101, as viewed by co-ip (Fig. 3 and4) and by FLIM-FRET (Fig. 6 and 7). FLIM, a method of choice to study protein-protein interaction [START_REF] Sun | Forster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells[END_REF][START_REF] Richert | Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy[END_REF] allows the monitoring of the fluorescence lifetime of eGFP-TSG101 in presence of Gag-TC-ReAsH as acceptor. Using this setup, analysis of cell expressing Gag, GagZF1 or GagZF2, using a two exponential analyses, showed that the distance between Gag and TSG101 was 54±0.1 Å. This short distance is independent from the deletion of a single zinc finger and from the site of cellular distribution of the complex in the cell (Fig. 6B, bluish color, upper raw), as shown by homogeneous distribution of  1 in the cytoplasm, in dots or at the PM.

Interestingly, amplitudes associated to the short-lived lifetime in the cytoplasm, in dots and at the PM (fig 6B, orange color, bottom raw) is highly similar to the co-localization between Gag, GagZF1 or GagZF2 and TSG101, as observed by confocal microscopy (figure 2 and5). All these results, suggests that Gag is the main driving force for TSG101 trafficking, but not viceversa.

A slight but statistically significant increase of 1 was observed on cells expressing GagZF1ZF2 and GagNC as compared with the lifetime obtained for Gag (Fig 7A). But, in comparison with the total absence of FRET for Gagp6, this result also shows that, in the context of Gag, the PTAP domain remains essential for interacting with TSG101. Nevertheless, the main impact of NC deletion or of the simultaneous deletion of both zinc fingers on Gag-TSG101 interaction lies in the important decrease in the population of Gag engaged in the Gag-TSG101 complex (Fig. 6B, panel l and m and Fig. 7B). This shift in population could be attributed to the decrease of Gag-TSG101 affinity thus suggesting that the NC domain of Gag strengthen the weak affinity determined for p6-TSG101 (Pornillos, Alam et al. 2002;Im et al. 2010). In accordance with this hypothesis, the NMR structure of NCp15 showed an inter-protein interaction between NCp7 and p6 with a close proximity between the central domain of NCp7

and the acidic residues of p6 (Wang et al. 2014). Since this fold back conformation interferes with the accessibility of NCp9/p6 by the protease (Wang, Naiyer et al. 2014), it may be hypothesize that PTAP motif of p6 domain is brought in a close proximity to the zinc fingers for promoting the recognition of NCp7 and p6 by TSG101. Furthermore, this dual-binding may results in compactness and stability of TSG101/Gag complex.

In addition, GagNC participates to the oligomerization of Gag and its assembly at the PM by virtue of NC interacting with the viral RNA and with membranes (Ott, Coren et al. 2009;[START_REF] Chukkapalli | Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain[END_REF]Muriaux and Darlix 2010;[START_REF] Kerviel | Virus assembly and plasma membrane domains: which came first?[END_REF]Kempf et al. 2015).

Nevertheless, these functions are independent of TSG101 recruitment, in accordance to the results obtained with G2A mutant that despite a defect in the Gag oligomerization and membrane binding co-localizes efficiently with TSG101 (Fig. S3A) and showed a FRET similar to wild type Gag (Fig. S3B). Moreover, the removal of RNA does not abrogate Gag-TSG101 interaction (Fig. 3C), which is in agreement with our results showing that basic residues surrounding zinc fingers are not directly involved in this interaction.

Interestingly, this NC-TSG101 interaction seems highly disparate as to the interaction between NC and Bro 1, occurring during the LYPXnL pathway (Popov et al. 2008;Dussupt et al. 2009). In fact, NC-Bro1 complex is sensitive to mutations in the basic domain of NC and also needs RNA that could bridge the two proteins [START_REF] Bello | Budding of retroviruses utilizing divergent L domains requires nucleocapsid[END_REF]. This brings to a hypothesis which implies that the two mechanisms, Gag/TSG101 and Gag/ALIX, maybe not redundant but concordant, in a way that the NC could recruit simultaneously both TSG101 and ALIX proteins in order to best optimize the chances of the virus to bud. Studies are currently underway to identify a possible simultaneous recruitment of these two cellular partners by the NCp15 domain of Gag and to define the kinetics of this interaction in living cells.

In line with our results, it was recently shown by in vitro GST pull down assay that NC is essential for p6 in recruiting TSG101. Our results extend this result by showing that, in the context of Gag and in cell expressing NC, the GagNC directly interacts with TSG101. Moreover, the group of Mougel et al also pointed out, using the duolink approach, that the second zing finger was important for the PTAP mediated budding of the virus (Chamontin et al. 2015). Using this technique, which is also referred as Proximity Ligation Assay [START_REF] Soderberg | Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay[END_REF], they showed that the second zinc finger is important for the co-localization of Gag and TSG101 at the PM and in particles. Nevertheless, the distance between two proteins emitting PLA signal, was estimated to 30-40 nm [START_REF] Soderberg | Direct observation of individual endogenous protein complexes in situ by proximity ligation[END_REF]) that hampered the assertion related to the direct interaction between the two proteins. On the contrary, using our approach of FRET we investigated more deeplyand determined that the second zinc finger was directly involved in the interaction of the two partners.

Data reported in this study shows a good correlation between experiments carried out by co-IP and by FLIM-FRET, indicating that there is a direct interaction between Gag and TSG101, that relies at least in part, on the NC region. However, one caveat between IP and FRET is when NCp7, transiently expressed in cells, was able to precipitate TSG101 while neither NC-mCherry nor NC-TC was found having FRET with eGFP-TSG101. Such a discrepancy between IP and images is probably due to the use of a surfactant that partially destroys the cell compartments.

Nonetheless our results reveal that NCp7 can potentially interact with TSG101.

In summary, we have used a series of biochemical assays to underline the role of NCp7 domain of Gag for the recruitment of TSG101. These approaches have provided evidences that the complex formation of Gag-TSG101 get compacted by direct interaction of TSG101 with GagNC and by the virtue of GagNC, Gag trafficking is responsible for the docking of TSG101 to the PM. Lysates of HeLa cells expressing HA-TSG101 alone or together with NCp7-eGFP or NCp7-eGFP derivatives were subjected to immunoprecipitation assay using @eGFP antibody and protein A magnetic dynabeads. Hela cells were transfected by plasmids expressing HA-TSG101

Legends

(well 1) with NCp7-eGFP (well 2), or NCp7ZF1-eGFP (well 3) or NCp7ZF2-eGFP (well 4)

or NCp7ZF1ZF2 (well 5). The total cytoplasmic extract was probed by mouse anti-eGFP and mouse anti-HA (input). Immunoprecipitation was performed with rabbit anti-eGFP followed by a western blot analysis with mouse anti-HA (IP). Membrane was reblotted with mouse anti eGFP to confirm the presence of NCp7-eGFP in immunoprecipitates. Note that the staining of HA-TSG101 is highly similar to the detection of eGFP-TSG101. with an excitation at 488nm or 561nm for eGFP and ResAsH respectively. Images correspond to the superimposition of the red and green channels. Gag-Gag interactions in a cellular context. It was observed that the FLIM approach provides a much better contrast than that based on epifluorescence microscopy. We managed conditions to visualize the sub-structures, at the PM and in the cytoplasm, where Gag assembly takes place, by FLIM. The importance of the work become more in magnitude as the conditions were characterized in for live cells (publication 1).

Figures:

Later on, we applied vigorously these quantitative fluorescent microscopy and biochemical approaches and we conclude mainly on two undermentioned aspects of budding process.

Gag-Gag Oligomerisation

To study Gag-Gag interaction, plasmids expressing Gag and mutations in Gag like GagΔNCp7, GagΔZF1, GagΔZF2 and GagΔZF1/ZF2 were transiently transfected and expression of proteins were followed by FLIM based FRET and western blot. By FRET, we were able to follow Gag-Gag interaction at the level of the plasma membrane in agreement with the role of this protein during the formation of the viral particles. Interestingly, FRET was also observed within the cytoplasm suggesting that the polymerisation of Gag is initiated before the anchorage of Gag to the PM. When NCp7 or both Zing fingers of NCp7 were removed from the Gag precursor, the Work is now in progress to describe the interaction between Gag and the RNA. Schematically, Gag interacts none specifically with cellular RNA during the assembly of the particle while a specific recognition between Gag and the viral RNA is required to encapsidate the genome.

Concerning the first issue, the RNA of cell expressing Gag/Gag-eGFP will be labelled with Sytox, a chemical compound that interacts both with RNA and DNA. This chromophore was used in the lab to monitor the interaction between NCp7 and cellular RNA by FRET (Anton, Taha et al. 2015) . If a direct interaction is evidenced between Gag-eGFP and RNA-Sytox by FLIM, we will use this tool to detail Gag-RNA interaction in cellular context. First, the role of GagNC in the recognition of cellular RNA will be assessed by comparing wild type GagNC and all the constructs deleted from the zinc fingers. It can be hypothesised that GagΔZF1ZF2, containing the basic residues surrounding the zinc fingers, will interact as well as wt Gag with cellular RNA.

Meanwhile, the relationship between Gag oligomerisation and Gag-RNA interaction will be tested using Gag mutated at the level of the CA. It was shown by us and others that GagWM could oligomerize by forming less dense structure. The level of FRET between GagWM and RNA Sytox will document the accessibility to the solvent for GagNC.

At last, the impact of the anchoring of Gag at the PM to the RNA recognition will be tested using Gag G2A construct or mutation in the HBR sequence of MA. In fact, in the actual model of Gag assembly, both MA and NC interact with the RNA and this Gag-RNA complex binds to the cytoplasmic leaflet of the PM. If the FRET varies with these constructs that would suggest that docking of Gag to the PM reorganises Gag-RNA interaction.

Concerning a specific interaction between Gag and RNA, a series of plasmids are currently under construction or obtained from the literature. Concerning the RNA, the construct contains all cis acting sequences required for the RNA to be export outside the nucleus (RRE) and encapsidated (Leader region encompassing the first 5-700 bases). In addition, this construct contains a series of stem loop interacting either with labelled MS2 or Lambda. This construct will be co-transfected with a plasmid expressing all proteins except ENV together with labelled Gag. The interaction between Gag and  containing RNA will be followed by FRET and/or by TIRF. Then mutations in cis acting and trans acting sequences will detail the mechanism whereby the specific GagNC is able to enrich the genomic RNA in nascent particles.

Gag trafficking/Gag-TSG101 interaction

After defining the vital role of NCp7 in Gag-Gag oligomerization and irreplaceable role in Gagtrafficking to the PM, we explored the interaction of Gag and, role of NCp7 domain, with other cellular proteins that may be involved during viral egress. Earlier studies with western blot technique showed that cells expressing GagΔNC resulted in decrease of viral particles production. Consistent with the role of NC region and ESCRT protein TSG101 in virus release, we investigated the interaction between TSG101 and Gag that constitute our second conclusion.

We validated the interaction between native Gag and Gag mutants like GagΔNCp7, GagΔZF1/ZF2, GagΔZF1, GagΔZF2 and GagΔp6 with TSG101, their co-localisation in cells, and the impact of Gag mutations on these assembly processes by immunopricipitation, confocal and FLIM microscopy technique. We demonstrated that the NCp7 alone or as a domain of Gag (GagNC) was required for Gag to interact with TSG101. Our results showed a drastic decrease ( 4to 6-times less as compare to wild type Gag) in binding and complete abruption of trafficking for Gag/Tsg101 complex in absence of NC domain. This indicates a reduced interaction between the two proteins when NCp7 domain is not present in the Gag precursor. Further experiments with NC mutants showed that the NCp7 mediated interaction between Gag and TSG101 necessitates at least one of the two zinc. Later on, by analyzing amplitudes associated to the short-lived lifetime in the cytoplasm, in vesicles and at the PM, it was concluded that Gag is the main driving force needed for TSG101 trafficking. As NCp7 region drives the trafficking of Gag and Gag drives the trafficking of TSG101, it is evident that NCp7 region is a must for TSG101

trafficking. Later that GagGag/TSG101 interaction is also confirmed in the context of whole virus by our collaborators in CPBS Montpellier.

Collectively, our results first support a model in which NCp7 domain cooperates with the p6 domain to interact with Tsg101, thus giving an insight into the functional link between p6 Late domain motifs and NC as well as regulatory role of NCp7 domain in the course of recruiting components of the cellular machinery necessary for HIV-1 budding. Moreover, our results also showed the RNA-independency of Gag-TSG101 interaction that seems highly contrasted to the interaction of NC and Bro 1 domain of ALIX protein during the LYPXnL pathway. This leaves to imply that the two mechanisms maybe not redundant but rather that the NC could recruit simultaneously both TSG101 and ALIX proteins in order to best optimize the chances of the virus to bud (Publication 3).

In the end, the regulatory effect of NCp7 domain in either TSG101 or Gag or both proteinregulated pathway during virus budding can be exploited to develop inhibitors targeting HIV-1.

Meanwhile, this information can be utilized to investigate temporal and spatial organization of Gag with respect to ESCRT proteins during HIV-1 budding, either by using invitro biophysical techniques or by super-resolution imaging. Another, key aspect of budding process can be explored that involve possible interactions of Gag with fission related proteins like dynamin 2, synaptojanin, endophilin, etc, by using this invitro approach.

Etude du trafic intracellulaire de la protéine Gag du VIH et rôle de son domaine NCp7. Puis, la phase symptomatique qui se Les trois phases de l'infection par le VIH-1 sont représentées par l'évolution de la charge virale plasmatique (quantité de virus dans le plasma, en rouge) et du nombre de lymphocytes T CD4+ circulants (en bleu) au cours du temps.

manifeste de 1 à 10 ans après la contamination. Si on mute une des histidines par une cystéine, cela entraîne une déformation de la structure des deux doigts de zinc qui se traduit par la production de virus non infectieux (7-10,11 ,12). Plus récemment, il a été montré que dans la phase tardive du cycle la NCp7, au sein du précurseur Gag (appelée ci-dessous GagNC), possédait aussi un rôle essentiel dans l'assemblage (pour revue (48)). En effet, les parties basiques de la protéine sont indispensables pour la formation des polymères Gag-Gag et permettent également la reconnaissance 'non spécifique' d'un ARN (24 ,33,[45][46][47]49). De leur côté, les motifs dactyles seraient plus impliqués dans le trafic intracellulaire de Gag (50) et dans la reconnaissance des partenaires cellulaires lors du bourgeonnement (33,34 ). Enfin, des mutations de la NCp7 donnent lieu à une RT tardive suggérant que la NCp7 via son rôle dans les interactions Gag-Gag puisse contrôler de manière temporelle le déclenchement de la reverse transcription (51,52).

I

  express my gratitude to Dr. Ludovic RICHERT, Dr. Pascal DIDIER and Romain VAUCHELLES for their assistance on the imaging platform PIQ. Thanks to Dr. Eleonore REAL for her comments and rigorous scientific advice. I am grateful to Marlyse WERNERT and Ingrid BARTHEL for their availability and administrative assistance.

Figure 2 :

 2 Figure 2: Global prevalence of HIV-1 infection in 2013. Estimated prevalence among young and adults per country, at the end of 2013(WHO 2014).

Figure 3 :

 3 Figure 3: Typical course of HIV infection. Patterns of CD4+ T-cell decline (blue curve) and virus load increase (red curve) over the average course of untreated HIV infection; any particular individual's disease course may vary considerably.During the period following primary infection, HIV disseminates widely in the blood and lymphoid tissue; an abrupt decrease in CD4+ T cells in the peripheral circulation is often seen. An immune response to HIV ensures, with a decrease in detectable viremia. A period of clinical latency follows, during which CD4+ T cells counts continue to decrease, until they fall to a critical level below which there is a substantial risk of opportunistic infections[START_REF] Pantaleo | New concepts in the immunopathogenesis of human immunodeficiency virus infection[END_REF]).

Figure 4 :

 4 Figure 4: (A) General Features of the HIV-1 Replication Cycle and (B) Models for HIV-1 particle structure. During the early phase of replication (upper portion of the diagram), envelope glycoproteins on the surface of the virus bind to CD4 (black) and chemokine (red) receptors on the cell surface, triggering fusion of the viral and cellular membranes. During or after uncoating of the core (orange), the viral RNA genome is reverse-transcribed to proviral cDNA, which is transported to the nucleus and integrated into the cellular DNA by the viral integrase enzyme. Transcription during the late phase (lower portion of the diagram) produces the full-length viral RNA,which is capped at the 5′-end and polyadenylated at the 3′end by the cellular machinery and transported to the ribosomes. Translation of the unspliced RNA produces the viral Gag and, through an occasional read-through, Gag-Pol proteins, which assemble and bud to form immature particles. Proteolytic cleavage of Gag by the viral protease releases the matrix (MA, green), capsid (CA, pink) and nucleocapsid (NC, blue) proteins, which rearrange to form the mature particle[START_REF] Lu | Structural determinants and mechanism of HIV-1 genome packaging[END_REF]).

Figure 5 :

 5 Figure 5: HIV-1 genome organization and virion structural features.Above, the HIV-1 genome encodes 3 main genes: gag, pol, and env, as well as the accessory proteins vif, vpu, vpr, tat, rev, and nef. The RNA transcripts that give rise to these proteins are generated as unspliced (gag and pol), singly spliced(env, vif, vpr, vpu) or multiply spliced (tat, rev, nef). For tat and rev, the 5' and 3' exon splice junction sites are marked by a bold line. Sequences found at both the 5' and 3' ends of the genome contain the long-terminal repeats (LTRs), regions that dictate viral RNA synthesis and integration, while genome packaging into new progeny virions is directed by the packaging signal psi at the 5' terminus of the transcript. The HIV-1 genome in this illustration is based on the HXB2 isolate. Below, a schematic of the structural features of the two virus forms: immature (left) and mature (right). The immature virus is characterized by a thick layer of uncleaved gag polyproteins underlying the virion membrane, whereas proteolytic processing of the gag polyproteins during maturation gives rise to a cone-shaped core that encloses the viral RNA and additional viral proteins. This processing step renders the virus infectious. Adapted from (Ganser-[START_REF] Ganser-Pornillos | The structural biology of HIV assembly[END_REF]).

Figure 6 :

 6 Figure 6: Illustration of the 5' LTR of proviral DNA integrated into the host genome. The LTR is divided into three regions U3, R and U5. The transcription starts at the beginning of the region R and the binding sites recognized by different transcription factors of RNAP II. These sites are represented upstream and are characterized into three regions: the "core" of the promoter (TATA box), the activator domain and the modulator domain(Ekram W. Abd El-Wahab, Redmond P. Smyth et al. 2014).

Figure 7 :

 7 Figure 7: Secondary structure of the translated region 5 'of the MAL isolate of HIV-1 (adapted from[START_REF] Berkhout | Structure and function of the human immunodeficiency virus leader RNA[END_REF]).

Figure 8 :

 8 Figure 8: Landmarks of the HIV-1 genome. Genetic organization of HIV-1 genome. Positions of 5' Cap, 3' polyadenylation tail, LTRs and RRE are indicated; ORFs are shown as rectangles. (Paillart, Shehu-Xhilaga et al. 2004).

Figure 9 :

 9 Figure 9: Structure of envelope proteins of HIV-1 (Caffrey 2011; Guttman, Kahn et al. 2012).HIV-1 Membrane proteins oligomerize in heterotrimeric. The three-dimensional structures represent a SUgp120 surface subunit of HIV-1 of one hand and the ectodomain of the transmembrane subunit under TMgp41 VIS in other hand.

Gag

  and Gag-Pol polyprotein precursors during virion maturation. The functional form of the protease enzyme is an asymmetrical homodimer stabilized by four-stranded antiparallel β-sheets formed by N-and C-terminal β-strands connected to each other by a β-turn and forms a flexible flap on top of the catalytic site (Figure 10)(Das, Prashar et al. 2006).

Figure 10 :

 10 Figure 10: Crystal structure of HIV-1 protease. Structure of the HIV-1 protease in its functional form. The catalytic site composed of two Asp residues is shown in blue. In the binding pocket, there is a polypeptide substrate shown in green.[START_REF] Lee | The choreography of HIV-1 proteolytic processing and virion assembly[END_REF]).

  the process of reverse transcription to convert the viral genome into a doublestranded DNA. The RNase H activity removes the original RNA template from the first DNA strand to allow the synthesis of the complementary DNA strand. However, RT has high error and mutation rates during synthesis of proviral DNA due to absence of 3'→5'exonucleolytic proofreading activity[START_REF] Hu | HIV-1 reverse transcription[END_REF].

Figure 11 :

 11 Figure11: The structure of the HIV-1 RT catalytic complex. The polymerase active site is on the left and the RNase H domain on the right. The domains of p66 are in color: fingers (red), palm (yellow), thumb (orange), connection (cyan), and RNase H (blue); p51 is in gray. In the two chains, the domains have very different relative orientations. The DNA template strand (light green) contains 25 nucleotides, and the primer strand (dark green), 21 nucleotides. The incoming dNTP is in gold[START_REF] Huang | Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance[END_REF]).

Figure 12 :

 12 Figure 12: Architecture of the HIV-1 intasome. (A) The inner subunits of the IN tetramer, comprising residues 1-270 and engaged with viral DNA, are green and cyan; outer IN CCDs (residues 56-202) are blue and yellow. The reactive and non-transferred DNA strands are magenta and orange, respectively. Red sticks, side chains of inner monomer active site residues; red and gray spheres, Mn and Zn ions, respectively. (B) Resection of the upper IN dimer from A highlights the part of the model analogous to the PFV crystallographic asymmetric unit. Locations of canonical IN domains are indicated (Cherepanov, Maertens et al. 2011).

  Figure 13: Overall structure of the Tat/PTEFb/ATP complex. (a)Ribbon representation and (b) surface representation of the Tat/PTEFb-ATP structure.Cdk9 is light orange, Cyclin T1 is pale green and Tat is magenta. The side chains of the Cdk9-interacting residues of Tat, Cys261 of Cyclin T1, and ATP analog are drawn as sticks, and the zinc and magnesium atoms are drawn as cyan and light blue spheres, respectively. The dashed lines represent the missing link between Lys88 and Gly35 of Cdk9 and between Leu252 and Cys261 of Cyclin T1[START_REF] Tahirov | Crystal structure of HIV-1 Tat complexed with human P-TEFb[END_REF]

Figure 14 :

 14 Figure 14: Structure of the Rev Dimer. (a) The folded core of a Rev monomer with its functional domains is shown. The amino acids prominent in mediating the core structure are specified. The different colors of the ribbon indicate amino acid conservation among 1201 HIV-1 isolates in the Los Alamos Sequence Database, with green representing the least conservation (26%) and red the highest (100%). (b) Model of the Rev dimer interacting with a model Rev Response Element (RRE) stem IIB binding site (Hammarskjold 2011).

Figure 15 :

 15 Figure 15: Three-dimensional structure of the complex formed between the Nef protein of HIV-1 and AP-2.Nef (orange) binds to the alpha subunits (cyan) and sigma 2 (magenta) of AP-2.[START_REF] Ren | How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4[END_REF].

Figure 17 :

 17 Figure 17: Schematic representation of the structure of the HIV-1 Vpu protein of[START_REF] Bour | The HIV-1 Vpu protein: a multifunctional enhancer of viral particle release[END_REF]. Vpu has a transmembrane domain (TM) and a cytoplasmic domain composed of two regions arranged in alpha helices.

Figure 18 :

 18 Figure 18: (A) Solid surface of Vif showing a negatively charged concave patch. Coloring is according to the electrostatic potential at the surface calculated with DELPHI. (B) 3D structure of VIF from the same viewpoint as (A); a three-dimensional model of VIF was constructed by comparative modeling based on two templates, VHL and NarL, which were used to construct the C-terminal domain and N-terminal domain of Vif respectively[START_REF] Lv | Three-dimensional structure of HIV-1 VIF constructed by comparative modeling and the function characterization analyzed by molecular dynamics simulation[END_REF]).

Figure 19 :

 19 Figure 19: Steps in HIV-1 proteolytic maturation, and variants analyzed. A) Schematic outline of the proteolytic cleavages which take place in Gag during the HIV-1 maturation process. Arrowheads indicate proteolytic sites before cleavage. The order of cleavage events shown is based on the rates of cleavage in vitro as described in (Pettit, Moody et al. 1994). B) Schematic representation of Gag after completion of cleavage for each variant analyzed. The non-cleaved products due to the inactivation of the proteolytic sites are highlighted. Mutated and therefore uncleaved processing sites are indicated by an arrowhead (de Marco, Muller et al. 2010)

Figure 20 :

 20 Figure 20: Schematic representation of Gag and its constituent domains. (A) Positions of myristoylated matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, and the spacer peptides SP1 and SP2 within the uncleaved Gag are indicated. (B) MA. Domains contributing to virus assembly, membrane binding, Gag targeting, post-entry steps, and Env incorporation are shown. The region of MA which forms the main globular domain is indicated, the position of the highly basic domain near the N terminus is shown. (C) CA. The N-terminal ''core'' and C-terminal ''dimerization'' domains are indicated. The Cyp-A binding loop and MHR are shown. (D) NC. The amino acid sequence is indicated and the two zinc-finger domains are shown. (E) p6. The two currently recognized major functional domains are shown: the N-terminal region involved in virus release and the C-terminal Vpr-interacting sequence. Amino acid positions are shown over each diagram. Adapted from (Adamson and Freed 2007).

  Figure 21: A. Three-dimensional structure of HIV-1 MA monomer and trimer as determined by X-ray crystallography. The putative membrane binding surface of the trimer is shown. B. Model of membrane binding by MA. 1: the NH2-terminal myristic acid moiety (green) of MA is depicted in its sequestered conformation. PI(4,5)P 2 embedded in the inner leaflet of the PM lipid bilayer is shown with its (orange) and (yellow) acyl chains in the lipid bilayer. 2: binding between MA and PI(4,5)P 2 leads to the flipping out of the myristate moiety into an exposed conformation with its subsequent insertion into the lipid bilayer, and the extrusion of the 2′acyl chain from the lipid bilayer and its sequestration by MA. 3: during the formation of the complex between HIV-1 MA and PI(4,5)P 2 , the 2′-unsaturated acyl chain of PI(4,5)P 2 (yellow) binds to the hydrophobic cleft in MA and the myristyl group (green) of MA inserts into the lipid bilayer (Balasubramaniam and Freed 2011).

Figure 22 :

 22 Figure 22: The architecture of the mature HIV-1 core. (a) Pseudo atomic model of an HIV-1 core showing the fullerene cone architecture, with the 12 pentamers highlighted in yellow. (b) Structure of the hexamer formed by the N-terminal domains of CA, showing the 18-helix bundle. (c) A higher magnification of the boxed region in(a) illustrating the6-fold interactions of the N-terminal domains (orange) and the 2-fold interactions of the C-terminal domains (blue) (Briggs and Krausslich 2011; Yeager 2011).

  Figure 23: (A) X-ray crystal structure of HIV-1 CA. Helices N1-N7 in the CA-NTD and C1-C4 in the CA-CTD, the CyPA-binding loop (red) and inter domain linker (green) are indicated (Mascarenhas and Musier-Forsyth 2009). (B) 3-D structure of the CA-CA dimer formed upon proteolytic cleavage of the MA-CA junction. The aminoterminal βhairpin, which forms only after proteolysis, mediates CA-CA interactions facilitating capsid core condensation during virus maturation (Yeager 2011).

Figure 24 :

 24 Figure 24: Structure and sequence of the HIV-1 NCp7 (adapted from (Godet and Mely 2010; Godet, Boudier et al. 2012). (A) Three-dimensional structure (PDB 1AAF) and (B) Sequence of NCp7. The zinc fingers (ZF) are shown in green, the basic linker is shown in orange and the N-ter domains and unstructured C-ter are shown in gray and magenta, respectively. (C) Identification of variables and conserved residues of NCp7 of HIV-1, resulting in the alignment of 3120 sequences base data HIV LANL (http://www.hiv.lanl.gov/).

Figure 25 :

 25 Figure25: Gag-Gag interaction monitored by FLIM. HeLa cells were transfected by a plasmid encoding for Gag-TC(Rudner, Nydegger et al. 2005). 24 hours post-transfection, cells were labeled with the FlAsH derivative(Fritz, Dujardin et al. 2010). Then, two-photon FLIM images were recorded and measured lifetimes were imaged using an arbitrary color scale. (top of panel 1). Panel 1, control naive HeLa cells incubated with FlAsH. The chromophore is homogeneously distributed with an average lifetime of 3.52 ns. Panel 2: HeLa cell expressing Gag-TC. The close proximity of Gag-TC-FlAsH molecules decreases their fluorescence lifetime to 2.52 ns, reflecting Gag multimerization. Gag-containing dots with an intermediate lifetime (green color) might correspond to Gag oligomers. Panel 3: Substitution of methionine 369 in the SP1 spacer for alanine (GagM369A-TC) severely impairs Gag assembly, as shown by the 3.08 ns fluorescence lifetime value obtained for this mutant. Panel 4: HeLa cell transfected with the GagG2A-TC mutant. The substitution of Gly for Ala hampers membrane anchoring of Gag. The fluorescence lifetime homogenously distributed throughout the cytoplasm is equal to 2.87 ns indicating that myristylation-defective Gag can oligomerize but does not form high molecular weight structures. This figure is adapted from fig 3 of (Hugues de Rocquigny* 2012)

Figure 26 :

 26 Figure26: VLP assembly results from the recruitment of cytosolic Gag clusters. Photoconversion scheme and intracellular localization of Gag-EOS chimera proteins. The green spheres correspond to the non-photoconverted form of Gag-EOS (exc= 488 nm and em=516 nm) observable in the epifluorescence mode. The red spheres correspond to the photoconverted form of Gag-EOS (exc= 516 nm and em=581 nm) after a photoconversion pulse at 405 nm in the TIRF mode. Gag coming from the cytoplasm (II) will be detected in the green channel while Gag already anchored at the plasma membrane (I) prior to the 405 nm pulse will be detected in the red channel. This figure is adapted from fig 3 of (Hugues de Rocquigny* 2012).

Figure 27 :

 27 Figure 27: Proposed mechanism for NC-promoted binding of Gag to the plasma membrane. (a) Newly made Gag polyprotein binds to the gRNA via its NC and MA domains, so that it adopts a bent conformation. (b) The Gag-gRNA complex also interacts with the plasma membrane via its MA and NC domains. (c) The NC domain recruits negatively charged lipids, including PI(4,5)P 2 , that form an acidic lipid-enriched microdomain. (d) The MA domain recognizes PI(4,5)P 2 , which results in the exposure of the previously sequestered myristyl group that inserts into the cytoplasmic leaflet of the PM. As a consequence of this anchoring of MA into the PM, MA dissociates from the gRNA. (e) Without the support of MA, the bent conformation is released and the Gag molecule adopts a rod-shaped conformation (Kempf, Postupalenko et al. 2014).

  specific and selective mechanism and has been shown to involve direct binding of packaging signal () and the nucleocapsid domain (NC) of the retroviral Gag precursor[START_REF] Berkowitz | RNA packaging[END_REF][START_REF] D'souza | How retroviruses select their genomes[END_REF][START_REF] Lever | HIV-1 RNA packaging[END_REF][START_REF] Lu | Structural determinants and mechanism of HIV-1 genome packaging[END_REF]. It is interesting to know that, despite the relative scarcity of the unspliced viral RNA (usually <1% of total RNA), it is strongly favored over abundant cellular RNAs and viral spliced mRNA species. Moreover, unspliced RNA is more efficiently packaged compare to spliced RNA despite a number of common packaging signals shared by the two RNAs namely: TAR, Poly-A, PBS and the DIS[START_REF] Paillart | Dimerization of retroviral RNA genomes: an inseparable pair[END_REF][START_REF] Lu | Structural determinants and mechanism of HIV-1 genome packaging[END_REF].Recently, Ekram W et al. demonstrated that the primary Gag binding site on the gRNA consists of the internal loop and the lower part of Stem-loop 1 (SL1). Along with, a double regulation by the upstream and downstream nucleotide sequences of SL1 ensures specific binding of Gag to the gRNA. The region upstream of SL1, which is present in all HIV-1 RNAs, prevent binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA[START_REF] Ekram | Specific recognition of the HIV-1 genomic RNA by the Gag precursor[END_REF]).

  incorporated into the particle during assembly namely, cellular tRNA 3 Lys (50-100 molecules/virion) (Isel, Ehresmann et al. 2010), several viral proteins including: Vpr (100-200 molecules/virion), Vif (10-100 molecules/virion), and Nef (5-70 molecules/virion). A number of cellular proteins are incorporated into viral particles including: Cyp-A (~250 molecules/virion), Hsp70 present at 1:25 Gag molecules, LysRS, TSG101, Ubiquitin, APOBEC3G, INI1/HSNF5,Staufen, UNG2, HLA-II, ICAM-1 (CD54), annexins and tetraspanins[START_REF] Coffin | Retroviruses Cold Spring Harbor Laboratory Press[END_REF][START_REF] Ott | Cellular proteins detected in HIV-1[END_REF][START_REF] Balasubramaniam | New insights into HIV assembly and trafficking[END_REF]. Furthermore, the interaction between the GagNC and the packaging domain of the gRNA allows further contacts between the viral RNA and tRNA Lys3 /LysRS facilitating thereby the annealing of the tRNA Lys3 to the PBS[START_REF] Kaminska | Viral hijacking of mitochondrial lysyl-tRNA synthetase[END_REF][START_REF] Kleiman | Formation of the tRNALys packaging complex in HIV-1[END_REF][START_REF] Kobbi | Association of mitochondrial Lysyl-tRNA synthetase with HIV-1 GagPol involves catalytic domain of the synthetase and transframe and integrase domains of Pol[END_REF]).

Figure 28 :

 28 Figure 28: HIV-1 budding and release. Recruitment of host factors by L-domains. Late-budding domains (Ldomains) encoded by enveloped viruses (grey boxes in the figure) recruits the ESCRT machinery through direct interactions with adaptor proteins in this pathway (red). Every known L-domain requires components of the core ESCRT machinery, such as ESCRT-III and vacuolar protein sorting-associated protein 4 (VPS4), to facilitate the final separation of virions from the infected cell. Auxiliary interactions that have been described HIV-1 Gag with the NEDD4-2 and NEDD4-2s isoforms and with Bro1 domain-containing proteins are shown (Marcello, Zoppe et al. 2001).

(

  [START_REF] Morita | Retrovirus budding[END_REF] Bieniasz 2009;[START_REF] Carlton | The ESCRT machinery: new functions in viral and cellular biology[END_REF][START_REF] Usami | The ESCRT pathway and HIV-1 budding[END_REF]) by mainly two and possibly three different pathways:

Figure 29 :

 29 Figure29: HIV-1 p6 mutants have a pronounced defect in release. Electron microscopy image of HIV-1 virions that carry mutations in p6 Gag. The membrane of both host cell and nascent virion remain connected through a thin membranous stalk. Adapted from(Gottlinger, Dorfman et al. 1991).

Figure 30 :

 30 Figure 30: Structure of TSG101 UEV and HIV-1 p6 peptide. (A) Schematic representations of human TSG101 and HIV-1 Gag. Abbreviations within TSG101 are: UEV (ubiquitin E2 variant) and SB ('steadiness' box). Viral protease cleavage sites are indicated by vertical lines and the names of resulting proteins are shown. The location of the PTAP peptide in p6 Gag is indicated. (B) Amino acid sequence and secondary structure of TSB101 UEV. The residues involved in PTAP peptide binding are colored in green. (C) Overall structure of TSG101 UEV and HIV p6 peptide. The Tsg101 UEV sequence consists of 145 amino acid residues. The location of β sheets and α helices are shown in the sequence and secondary structure. The position of the beta hairpin tongue is indicated. Residues in green are important for binding to the p6 PTAP peptide. Modified from (Im, Kuo et al. 2010).

Figure 31 :

 31 Figure 31: Model for GagNC cooperation with the PTAP/Tsg101 and LYPXnL/Alix budding pathways. Role of GagNC in the LYPXnL/Alix pathway: GagNC interacts with the Bro1 domain of Alix through its N-terminal basic residues and zinc fingers to recruit the essential downstream budding machinery components, ESCRT-III and VPS4, to promote virus egress.(Dussupt, Javid et al. 2009)

Figure 32 :

 32 Figure 32: Schematic representation of the essential core ESCRT machinery during budding. Late domain motifs within p6Gag binds directly to the UEV domain of the TSG101 subunit of the heterotetrameric ESCRT-I complex (red, with bound ubiquitin in black) and the V domain of ALIX (blue) to recruit the ESCRT-III proteins of the CHMP1, CHMP2, and CHMP4 families (green), which apparently polymerize into a "dome" that promotes closure of the membrane neck. Due to these interactions, VPS4 ATPases also get recruited that completes the membrane fission reaction and uses the energy of ATPase to release the ESCRT-III from the membrane and back into the cytoplasm (Sundquist and Krausslich 2012).

1 -

 1 The first aim of the thesis was to analyse the influence of the NC zinc finger (ZF) on the intracellular localization and assembly of HIV-1 Gag. Therefore, we used fluorescent Gag derivatives such as Gag-eGFP and Gag-mcherry, where the fluorophore was inserted next to the MA domain and monitored the assembly of Gag by the two photon fluorescence lifetime imaging microscopy (FLIM) based fluorescence energy transfer (FRET) and by the time laps epifluorescence microscopy. Detailed analyses of FRET images revealed a homogenous distribution of Gag-Gag complex in cells with a progressive concentration of ordered Gag oligomers underneath the plasma membrane (PM) favouring a model where Gag oligomerization in the cytoplasm precedes virus assembly at the PM. In this model, the absence of NC domain results in i) an accumulation of Gag as large aggregates that are dispersed in the cytoplasm, ii) a decrease of Gag-Gag condensation and iii) a delay for Gag-Gag complexes in reaching the PM. Thus GagNC is involved directly through protein-protein interaction or indirectly by its virtue to interact with RNA to the formation and the trafficking of Gag in the cell.2-Next, as a result of first aim of thesis, the monitoring of Gag assembly as function of time revealed a large decrease of VLPs at the level of the PM which is in agreement with data showing a defect in Gag, in the supernatant of cell expressing GagNC(grigorv 2007, hogue et al 2009). Taking into account the importance of TSG101 in Gag budding, our second aim was to determine the role of GagNC in the TSG101 pathway. For this, cells were transfected with HA-TSG101 together with plasmids expressing Gag or Gag mutants that are formed by deleting either the zinc finger motifs or the entire GagNC domain. Results were compared with cell co-expressing TSG101 and Gag-L6I7, in which PTAP sequence was substituted for LIAP[START_REF] Freed | The HIV-TSG101 interface: recent advances in a budding field[END_REF] or GagΔp6. Using a series of experiments including immunoprecipitation, confocal fluorescent microscopy and FLIM-FRET, we found that NC alone or in the context of Gag, interacts directly with TSG101 and could participate with p6 in the recruitment of ESCRT-I budding pathway of HIV-1. are the first immortal cell line of human origin and named after HEnrietta LAcks. Isolated in 1951, these adherent cells derived from metastases and were taken from a patient with cervix cancer.

  DH5α competent bacteria are prepared in the laboratory and were used to amplify plasmids. Several mutations are found in the genotype of the bacterium. Examples include mutations that endA1 and recA1 which respectively leads to inactivation of an intracellular endonuclease thus allowing greater protection of foreign plasmid DNA and allows the elimination of homologous recombination. The bacteria are stored at -80 ° C in TB buffer (10 mM Hepes, 250 mM CaCl2, 25 mM KCl, 55mM MnCl2, pH 6.7) containing 2% DMSO.

Figure 33 :

 33 Figure 33: Cell Neubauer (According www.celeromics.com)The area of the single square is denoted by 1mm 2 and the volume by 0.1 mm 3 = 1.10 -4 ml. Thus, to count the cells, the undermentioned formula was used:

  [START_REF] Azoulay | Destabilization of the HIV-1 complementary sequence of TAR by the nucleocapsid protein through activation of conformational fluctuations[END_REF] Clamme, Azoulay et al. 2003;[START_REF] Clamme | Intracellular dynamics of the gene delivery vehicle polyethylenimine during transfection: investigation by two-photon fluorescence correlation spectroscopy[END_REF][START_REF] Ludovic Richert | Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy[END_REF] consists of an inverted fluorescence microscope Olympus IX70 with a water objective lense (60X, Numerical aperture = 1.2). The fluorophores are excited by femtosecond pulsed laser (Insight Spectra Physics) which delivers pulses of 100fs between 680 and 1300nm. The fluorescence emitted by the sample is recorded with the avalanche Photodiode (APD) coupled to a TCSPC board (SPC 830). This system allows obtaining images in which the contrast is given by the lifetime of the fluorescence of eGFP and intensity images. The raw data is analyzed using commercial software (Image SPC, BH). A fluorescence lifetime image is generated by using a color scale reflecting the fluorescence lifetime by color (for example shorter lifetime in blue and longer lifetime in red) at each pixel. Thus a reconstruct of the observed cell life are done and allow the determination of the presence of interactions between two proteins exists or not. We considered that a minimum of 5% of FRET is needs to proof interactions between the two tagged proteins and exclude random interactions.

Figure 34 :Figure 35 :

 3435 Figure 34: Principle of the FRET-FLIM.

Figure 36 :

 36 Figure 36: 96 wells plate for a Bradford assay, For example, 3 samples the absorbance at 595nm is obtained by the spectrofluorimeter safas FLX-Xenius. The optical densities obtained are used to calculate the protein concentration of the samples and thus determine the amount of protein.

Figure

  Figure 37: The principle of co-immunoprecipitation

Figure

  Figure 38: Schematic representation of different mutations

Figure 39 :

 39 Figure 39: Thermocycler of PCR.

For

  live/fixed-cell imaging we also used TC-FlAsH/ReAsH expression tag-based fluorescence technique. It is ideal for protein localization or real-time protein production studies, though its versatility offers a range of benefits. FlAsH is a fluorescein derivative and contain two arsenic atoms at a set distance from each other. ReAsH is based on resorufin and has been similarly modified. Both on them are virtually nonfluorescent when bound to ethane dithiol (EDT). The biarsenical labeling reagents FlAsH-EDT2 and ReAsH-EDT2 become fluorescent after binding to the recombinant proteins containing the tetracysteine (TC) motif Cys-Cys-Pro-Gly-Cys-Cys (Figure 40).

Figure 40 :

 40 Figure 40: Mode of action of FlAsH and ReAsH

  Gag-Gag complex in cells with a progressive concentration of ordered Gag oligomers underneath the plasma membrane (PM) favouring a model where Gag oligomerization in the cytoplasm precedes virus assembly at the PM. In this model, the absence of NC domain results i) in an accumulation of Gag as large aggregates that are dispersed in the cytoplasm, ii) in lowering of Gag-Gag condensation and iii) in a delay for Gag-Gag complexes in reaching the PM. Thus NC is involved directly through protein-protein interaction or indirectly by its virtue to interact with RNA to the formation and the trafficking of Gag in the cell.

1 .

 1 5h. Beads were then washed three times with NP40 lysis buffer, denatured for 5 minutes at 95 ° C in Laemmli buffer and analysed by 12% SDS-PAGE. Membranes were incubated with mouse anti HA (Pierce, ref : 26183) or mouse anti-eGFP (Proteintech, France, ref : 66002-1-Ig) or mouse monoclonal anti Gag (Ref 6521 #24-4 ; AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. Michael H. Malim) or mouse anti-GAPDH (Millipore,MAB374, 1 :5000) followed by anti-mouse HRP (Promega, ref W402B). Proteins were visualized by the chemiluminescent ECL system (Amersham) on a LAS 4000 apparatus. Biarsenical labeling. HeLa cells cultured on 35-mm coverslips (μ-Dish IBIDI; Biovalley) were transfected with 1µg of plasmid encoding eGFP, or wild-type eGFP-TSG alone or together with

Figure 6 and 7 First

 7 Figure 6 and 7 First, cells expressing eGFP-TSG101 and labeled by ReAsH were imaged to determine the intrinsic fluorescence lifetime of eGFP-TSG101. The fluorescence decay of eGFP fused to TSG101was analysed using a mono-exponential model to calculate a mean fluorescence lifetime ( m ) of 2.26±0.07 ns. As a consequence, TSG101 staining appears as punctuated orange dots in the cytoplasm (Fig 6A, panel a). Moreover,  m is almost similar to that of free eGFP (2.39 ±0.08 ns, data not shown), suggesting that eGFP fluorescence was moderately modified due to its fusion to TSG101 or by the presence of the acceptor.

  long-lived lifetime  2 was fixed at the lifetime of eGFP-TSG101 (2.25 ns), while the short component  1 and the populations (α 1 and α 2 ) associated with the two lifetimes were allowed to float . Thus, it was possible to determine the short component and the associated fraction of eGFP-TSG101, in the complex. As depicted on Fig 6B, panel d, cell expressing eGFP-TSG101 and Gag-TC-ReasH were characterized by a blue color corresponding to an average  1 of 0.98±0.13 ns. Interestingly, this high FRET (56.3±8%, Fig7A) is homogeneously distributed all over the cell indicating that the tight interaction between the two proteins occurs in the cytoplasm, in dots (white arrows) and at the PM. The same analysis was carried out on cell expressing GagZF1, GagZF2, GagZF1ZF2 and GagNC (Fig 6B, panels e to h), showing the average  1 centered to 1.02±0.03 ns for GagZF1 and GagZF2 and to 1.18±0.04ns for GagZF1ZF2 and GagNC, respectively. Meanwhile, the amplitude 1 of the  1 component i.e. the fraction of eGFP-TSG101 species undergoing FRET, was found centred at 60% (Fig 7B) and was independent of the presence or the absence of any singular zinc finger. Thus, cells expressing

Figure 1 :

 1 Figure 1: Schematic representation of Gag and TSG101 fusion constructs.

Figure 2 :

 2 Figure 2: Co-expression of eGFP-Tsg101 and pNL 4.3 293T cells were co-transfected with viral DNA, eGFP-TSG101 and Gag-mCherry expressing plasmids (in a ratio 1/1). After 24h, cells were fixed with 4% PFA, cover slips mounted in presence of DAPI and observed by confocal microscopy. Images a to c: visualization of cell expressing of eGFP-TSG101 alone. Images d to f: visualization of cell co-expressing eGFP-TSG and Gag/Gag-mCherry. Images g to i: visualization of cell expressing eGFP-TSG101 and GagNC/GagNC-mcherry.

Figure 3 :

 3 Figure 3: Both zinc fingers of GagNC participates with p6 to recruit TSG101Lysates of HeLa cells expressing HA-TSG101 alone or together with Gag and Gag derivatives were subjected to immunoprecipitation assay using @HA antibody and protein A magnetic dynabeads. Hela cells were not transfected (well 1) or transfected by plasmids expressing HA-TSG101 (well 2) with Gag (wells 3), or GagZF1 (well 4) or GagZF2 (well 5) or GagNC (well 6) or Gagp6 (well 7) or GagZF1ZF2 (well 8). Well 1': control corresponding to beads incubated with a sample containing HA-TSG and Gag but without anti HA. Well 2': sample containing HA-TSG101 and Gag treated with RNAse prior to IP. A: total cytoplasmic extract (Input). B: immunoprecipitation using rabbit anti-HA followed by a western blot analysis using mouse anti HA. C: Blot B was re-probed with polyclonal anti-Gag to identify the presence of Gag in the immunoprecipitates. D: Ratio of Gag and Gag derivatives pulled down by eGFP-TSG101. Levels of grey corresponding to HA-TSG (membrane B) and to Gag (membrane C) were determined by Image J and normalized to the signal obtained in the supernatant containing

Figure 4 :

 4 Figure 4: Free NCp7 needs both zinc fingers to interact alone with TSG101

Figure 5 :

 5 Figure 5 : Co expression of eGFP-TSG101 and Gag or Gag derivatives by confocal microscopy Confocal microscopy of HeLa cells expressing eGFP-TSG101 (a) together with Gag/Gag-mCherry (b), or GagZF1/ GagZF1-mCherry (c), or GagZF2/ GagZF2-mCherry (d), or GagZF1ZF2/ GagZF1ZF2-mCherry (e), or GagNC/ GagNC-mcherry (f), or Gagp6/Gagp6-mCherry (g), or NC/NC-mCherry. For each Gag derivative, HeLa cells were transfected with 0.25µg of plasmid eGFP-TSG, 0.5µg of plasmid expressing Gag (or mutated Gag) and 0.25µg of plasmid expressing Gag-mCH (or mutated Gag-mCherry). Cells were fixed by 1.5% of PFA 24h post transfection, and analyzed by confocal microscopy. Each panel shows the major observed phenotype.

Figure 6 .

 6 Figure 6. Impact of NC deletion on Gag-TSG101 interaction, as monitored by FLIM. Hela cells were seeded on 35 mm coverslips and transfected with plasmids expressing eGFP-TSG101 with Gag derivatives (in a ratio 0.2 eGFP-TSG101 / 0.8 Gag). 24h post transfection, cells were washed and labeled with ReAsH. FLIM measurements carried out using a 50×50 µm scale and 128×128 pixels as settings. A: The fluorescence lifetime of eGFP-TSG101 (a) or eGFP-TSG101co-expressed with Gagp6-TC (b), or NC-TC (c) was measured in each pixel and

Figure 7 :

 7 Figure 7: Histograms presenting FRET efficiency and population of the complex betweenGag or Gag derivatives with TSG101A: Histograms representing the FRET efficiencies between TC-labeled Gag or Gag mutants and eGFP-TSG101. The FRET efficiency was calculated using the equation (1) with the average lifetime values from at least 30 cells in three independent experiments. B: Histograms representing population of TSG101 engaged in Gag-TSG101 complex. Student tests were performed to compare the FRET efficiencies and populations (*: p<0.05, ****: p<10 -3 ).

Figure S1 :

 S1 Figure S1: Gag mediated localization of either HA and eGFP-tagged Tsg101. HeLa cells were transfected with plasmid expressing HA-TSG101or eGFP-TSG101 together with a mixture of plasmids expressing Gag and Gag-mCherry. Cells were fixed with 1.5% of PFA. Panels a and b: cells were incubated with rabbit anti HA followed by anti-Rabbit Alexa 488. Panels c and d : cells were observed by confocal microscopy without immunodetection.

Figure S2 :

 S2 Figure S2: Gag-TC and Gag-TC derivatives detected by ReAsH HeLa cells were transfected with plasmid expressing eGFP-TSG101 or together with Gag-TC or Gag-TC derivatives. Cells were incubated with ReAsH and observed by confocal microscopy

Figure 7 General

 7 Figure 7

  average lifetime was intermediate suggesting a profound defect in Gag-Gag assembly. Moreover, mutated Gag was only partly accumulating to the PM showing a strict correlation between Gag oligomerization and Gag accumulation at the periphery of the cell. Our results clearly indicate that Gag proteins form compact oligomers in the cytoplasm, likely as a result of both CA-CA interactions and NC-promoted binding to nucleic acids. The oligomers progressively assemble, and grow in size during trafficking from the cytoplasm toward the membrane. The NC domain favors the compactness and binding of Gag oligomers to the PM and the proper timing of viral particle assembly at the PM, likely as a result of its interaction with cell proteins involved in Gag trafficking. While the proximal zinc finger appears dispensable for oligomer compaction and membrane binding, the distal finger and the flanking basic domains appear critical for these properties. Based on these results, the full NC sequence appears important for the proper timing of Gag assembly (Publication 2).

Figure 1 :

 1 Figure 1 : Evolution clinique de l'infection par le VIH-1.

  biochimiques telles que la myristoylation, la multimérisation, ou la phosphorylation. • La capside CA se subdivise en un domaine N-terminal (résidus 1 à 145) et un domaine C-terminal (résidus 146 à 231). Le domaine CTD est composée de 4 hélices alpha et une petite partie C-terminale de 12 acides aminés, très flexible n'adoptant pas de structure particulière.C'est ce domaine CTD qui assure l'essentiel de l'interaction Gag-Gag (3) requises lors de l'assemblage.• La protéine virale de nucléocapside NCp7 se compose sous sa forme mature de 55 acides aminés et présente un poids moléculaire de 7 kDa. C'est une petite protéine basique, caractérisée par deux motifs en doigts de zinc très conservés, CX2CX4HX4C, aussi appelés CCHC, connectés par une séquence basique. Ces doigts de zinc sont aussi appelés motifs dactyles et ils lient le zinc avec une très forte affinité (Ka = 10 -14 M -1 ) (4).

Figure 3 :

 3 Figure 3 : Structure primaire de la protéine de nucléocapside NCp7

I- 3 -

 3 La NC possède de nombreuses fonctions au cours du cycle viral. Une activité de chaperonne des acides nucléiques qui est essentielle lors de la transcription inverse, en induisant l'hybridation de l'ARNt Lys3 à l'ARN viral, en augmentant la vitesse de la réverse transcriptase et en activant fortement le saut des brins(13,14). Cette activité consiste à promouvoir le réarrangement des acides nucléiques vers leur conformation la plus stable en favorisant les duplex (15). Cette activité se décompose en trois étapes : interaction avec les acides nucléiques cibles, déstabilisation des brins et hybridation des brins complémentaires. La NCp7 permet aussi la protection de l'ARNg viral contre la dégradation par les nucléases et augmente également l'efficacité d'intégration de l'ADN viral dans la cellule hôte en permettant la formation d'un complexe[START_REF] Lapadat-Tapolsky | Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle[END_REF]. Enfin, NCp7 a un rôle très important dans l'encapsidation de l'ARN génomique. Elle présente une forte affinité pour les tige-boucles SL1,SL3 et SL4 (18,[START_REF] Clever | RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1[END_REF]. Elle est le médiateur de la reconnaissance de l'ARN viral parmi l'ensemble des acides nucléiques cellulaires. Les tige-boucles SL1, SL2, SL3 et SL4 ont chacune une affinité différente pour la NCp7 de telle manière que le site reconnu par la protéine est réparti sur les quatre tige-boucles (20) même si plus récemment il a été montré que la boucle SL1 contenait probablement un site de haute affinité (21). Les doigts de zinc sont nécessaires à l'encapsidation (22,23) mais n'ont pas la même importance. En général, toute mutation entraînant la déstructuration des doigts aura pour effet de diminuer l'encapsidation de l'ARN génomique au profit d'autres ARN notamment les ARN ribosomiques (23) (24). Mais le doigt à l'extrémité Nterminale joue un rôle plus important que l'autre doigt puisqu'il reconnaît l'ARN viral (25). Une mutation de ce doigt a pour effet de faire baisser considérablement l'affinité de la NCp7 pour la séquence SL3 (11). • Enfin, la protéine p6 qui se situe à l'extrémité C-terminale du précurseur polyprotéique Gag. Cette protéine mature se compose de 52 acides aminés et a un poids moléculaire de 5 kDa et est surtout impliquée dans le bourgeonnement des particules virales (26). Elle possède deux domaines appelés domaines tardifs ou 'late motif' qui interagissent avec plusieurs protéines de la famille des ESCRT. Ces protéines cellulaires sont impliquées dans la fission des membranes permettant 1-de fusionner des vésicules dans les MVB (multivesicular bodies) et 2-de séparer les cellules filles lors de la cytokinèse (revue dans (27)). Le premier motif tardif est une séquence riche en proline PTAP qui permet l'interaction avec le domaine UEV TSG101 du complexe ESCRT-1 (28-30). Le deuxième motif tardif qui semble moins important chez VIH-1 mais au contraire primordial chez d'autres rétrovirus est localisé dans la partie C terminal de p6. C'est la séquence YPXL qui recrute la protéine Alix et qui est en fait un co-facteur des protéines ESCRT (31,32). De manière intéressante cette protéine ALIX, notamment son domaine Bro est aussi reconnu par la NCp7 lors du bourgeonnement de la particule (33-35). Ces deux types d'interactions entre Gag et TSG101 ou ALIX permettent surtout à VIH-1 de détourner les protéines cellulaires de la famille des ESCRT à son profit. En effet, cette interaction permet le recrutement des domaines ESCRT-3 (notamment le domaine CHMP4) et de VPS-4. La protéine CHMP4 est une protéine héliocoïdale qui, en étant recrutée, va polymériser de manière spirale en créant un point de bourgeonnement (36). Cette polymérisation va aussi permettre l'interaction avec la protéine VPS4, qui est une hydrolase dépendante ATP et qui en hydrolysant les protéines du domaine ESCRT-3 libère le virus de la cellule infectée (37). L'ensemble de ce processus impliquant la protéine p6 est très dynamique et de nombreuses études notamment utilisant des outils modernes de microscopie de fluorescence ont montré que le recrutement des protéines ESCRT nécessitait environ 10 minutes (38,39) suivi d'environ 2 minutes pour libérer la particule sous l'action de la VPS4 (38 ,40). Rôle de Gag dans l'assemblage et le bourgeonnement viral: La polyprotéine Gag du VIH-1, est l'élément central pilotant la formation des particules virales (41). De nombreux travaux montrent que cet assemblage résulte d'une polymérisation de Gag impliquant deux plateformes. L'une est lipidique avec l'ancrage du myristate en N terminal du domaine MA de Gag dans une membrane cellulaire et l'interaction de ses acides aminés basiques avec les charges négatives des phosphatidylinositol 4,5-biphosphate de la membrane plasmique (MP) (42). L'autre plateforme est nucléique avec une interaction entre la (NCp7) de Gag et l'ARN génomique. Par ailleurs, les domaines de Gag indispensables à sa polymérisation sont localisés au niveau de la capside (région MHR (Major Homology Region) (43)) et dans le domaine N terminal de la NC (44-47).

1 -

 1 Les études sur l'assemblage de Gag puis son trafic et sa localisation à la MP aboutissant au bourgeonnement ont soulevé de nombreuses questions. Ainsi, le degré d'oligomérisation de Gag dans le cytoplasme et le mécanisme d'assemblage au niveau de la MP restent mal connus(33,53). Par ailleurs, l'assemblage a lieu principalement à la MP dans des cellules HeLa, Cos, 293T et T alors que dans les cellules de type macrophage et dendritique, il a lieu principalement au niveau de compartiments intracellulaires type LE (Late Endosomes)/MVB (Multi-Vesicular Body) (54). Cette localisation intracellulaire pourrait provenir soit d'un trafic par la voie endosomale de Gag soit de la MP après endocytose avant d'être dirigé vers une dégradation(55,56 ,57 ,58 ,59). Outre les différents types cellulaires et une éventuelle différence de détectabilité des différentes populations de Gag par immunofluorescence ou pas fusion à la GFP (60), le facteur temps pourrait être à l'origine de ces différences de voies. En effet, beaucoup d'expériences sont réalisées à un temps fixe post-transfection alors que des études en temps réel ont montré un parallèle entre la diminution de Gag dans des vésicules et l'augmentation de celleci à laMP (57,[START_REF] Sherer | Visualization of retroviral replication in living cells reveals budding into multivesicular bodies[END_REF]. Enfin, parallèlement à cet assemblage, la partie C terminale de Gag correspondant à la NCp15 (NC+p6) recrute des facteurs cellulaires (Tsg 101, ALIX) permettant la fermeture des membranes et le relarguage des particules (62) (Figure28).Les avancées techniques récentes dans le domaine de la microscopie à fluorescence ont permis de suivre la protéine Gag dans les cellules vivantes. La protéine fluorescente XFP (X= eGFP, mCherry, C, Y) a été placée soit en position C terminale soit en aval de la matrice. Dans ce dernier cas les propriétés d'assemblage et la fonctionnalité de la protéine sont proches de la protéine sauvage[START_REF] Muller | Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative[END_REF]. Ces études détectent des interactions Gag-Gag principalement au niveau de laMP (49 ,[START_REF] Larson | Visualization of retrovirus budding with correlated light and electron microscopy[END_REF][START_REF] Hubner | Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1[END_REF]. Par ailleurs, un tag tetra-cystéine (TC) a été placé aux mêmes positions ; la taille de cette séquence de 12 résidus étant compatible avec le suivi des particules dans les cellules[START_REF] Rudner | Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling[END_REF]. Cette séquence interagit de manière irréversible avec FlAsH ou ReAsH pour une émission de fluorescence respectivement dans le vert ou dans le rouge (67). Avec cette approche, le groupe de Freed montre que, dans les macrophages, les vésicules intracellulaires de Gag seraient plus particulièrement utilisées pour infecter une cellule saine par contact cellule-cellule (68). De plus, avec une approche similaire il a été confirmé, dans les cellules HeLa, que Gag utilisait la voie MVB/LE pour s'assembler (69). De façon tout à fait complémentaire, des études biochimiques de fractionnement sub-cellulaires de lymphocytes-T chroniquement infectés et d'immunofluorescence de cellules HT montrent également un possible assemblage de Gag intracellulaire(56,57). Tous ces résultats soulignent l'importance des modèles cellulaires utilisés pour étudier le trafic et l'assemblage de Gag.Enfin, des données récentes, utilisant une approche de microscopie TIRF (Total Reflection Internal Fluorescence) ont permis de suivre l'apparition/disparition de la particule virale au niveau de la membrane plasmique. Ces données, obtenues sur cellules HeLa, montrent qu'une première population de particules virales apparait/disparait avec des temps très courts (5-10 sec.) et leur co-localisation avec le marqueur d'endosomes CD63 suggère leur internalisation par endocytose (39). Une deuxième population est décrite avec une cinétique d'apparition de Gag de l'ordre de dix minutes suivie d'une période plus longue d'environ 25 minutes pouvant correspondre soit à un assemblage au niveau de la MP à partir d'une fraction cytosolique de Gag soit à une ré-internalisation de Gag dans le cytoplasme(38,39 ). Ainsi, cette étape de localisation de Gag à la membrane et de bourgeonnement pourrait être influencée par un équilibre entre le relargage extracellulaire de Gag sous forme de particule virale et son internalisation dans la cellule par endocytose.II-Projet de thèse :Le but de ma thèse a été de décrire le rôle de la NCp7 dans l'assemblage et le bourgeonnement de Gag dans la cellule en utilisant notamment la microscopie quantitative fluorescente. Au cours de mes études, j'ai construit mes expériences autour de deux objectifs :• Le premier objectif de la thèse est d'analyser l'influence du doigt de zinc NC (ZF) sur la localisation intracellulaire et l'assemblage du VIH-1. Par conséquent, nous avons utilisé des dérivés de Gag fluorescents tels que Gag-eGFP et Gag-mCherry, où le fluorophore est inséré à côté du domaine de MA. Puis la formation du complexe Gag-Gag a été caractérisée par FRET en suivant le temps de vie de fluorescence de l'eGFP par microscopie à deux photons. Des analyses détaillées des images FRET ont révélé une distribution homogène de complexe Gag-Gag dans les cellules avec une concentration progressive des oligomères Gag en dessous de la membrane plasmique (PM) favorisant un modèle dans lequel l'oligomérisation de Gag dans le cytoplasme précède l'assemblage du virus de la PM. Dans ce modèle, l'absence du domaine NC de Gag donne naissance à de gros agrégats qui sont dispersés dans le cytoplasme, à une diminution de la condensation de Gag et à un retard pour les complexes Gag-Gag à atteindre le PM. Ainsi NC est impliquée directement par l'interaction protéine-protéine ou indirectement par sa vertu d'interagir avec l'ARN à la formation et au trafique de Gag dans la cellule.• Au cours de ces premiers travaux, nous avons relevé une forte diminution de VLP au niveau de la PM qui, est en accord avec des données de la littérature, indiquait un défaut dans Gag, dans le surnageant de cellule exprimant GagNC. Tenant compte de l'importance de TSG101 dans bourgeonnement Gag, notre deuxième objectif de la thèse était de déterminer le rôle de la NC dans la voie de TSG101. Pour cela, les cellules ont été transfectées avec HA-TSG101 avec des plasmides exprimant Gag ou des protéines Gag mutées au niveau des motifs à doigts de zinc ou l'ensemble du domaine NC. En utilisant une série d'expériences comprenant une immunoprécipitation, microscopie à fluorescence confocale et FLIM-FRET, nous avons trouvé que NC seule ou dans le cadre de Gag, interagit directement avec TSG101 et peut participer avec p6 dans le recrutement de ESCRT-I du bourgeonnement du VIH-1. Suivi du complexe Gag-Gag en milieu cellulaire: Nous avons cloné les protéines Gag-eGFP, Gag-mCherry ainsi que de nombreux mutants des motifs CCHC (doigts de zinc ou motifs dactyles) du domaine NC de Gag. L'interaction Gag-Gag est suivie par FLIM à différents temps post transfection. Cette technique d'imagerie permet d'enregistrer le déclin de fluorescence de l'eGFP à chaque pixel de l'image (publication 1). Le traitement de ce déclin avec un modèle à deux composantes permet de calculer le temps de vie de fluorescence et la portion des protéines impliquées dans cette interaction. Une interaction protéine-protéine va induire du FRET qui va se traduire par une diminution du temps de vie de l'eGFP ( contrôle = 2.4ns). La figure ci-dessous montre un exemple d'images de FLIM 12h et 24h post transfection du couple Gag-eGFP/Gag-mCherry. Les images du haut correspondent à la répartition des temps de vie (Fig 1, image a :  m 1.4ns et image b  m 1.2ns) donnant un FRET d'environ 50% ce qui traduit un complexe Gag-Gag très compact. Les images du bas (c et d) décrivent les pourcentages de population de Gag qui forment des oligomères.

Figure 4 :

 4 Figure 4 : Assemblage de Gag à la membrane :

Figure 6 :

 6 Figure 6 : Le domaine NC de Gag peut avec le domaine p6 participer à l'interaction Gag-TSG101.A : analyse par western blot de l'input montrant que la TSG101 (anti-HA) et Gag (anti-Gag) et les mutants de Gag sont sensiblement exprimés de manière égale dans les différents échantillons les différentes constructions par rapport à la GADPH. B : l'anti-corps anti-HA précipite de manière similaire la protéine HA-TSG101. C : la membrane B est aussi révélée par l'anti Gag et montre que les différentes constructions de Gag ne sont pas toutes précipitées par la protéine TSG101. D : Histogramme montrant le niveau de précipitation de Gag par TSG101 en fonction des différentes constructions de Gag.

7 *Figure 7 :

 77 Figure 7 : Précipitation de TSG101 par la NCp7-eGFP Cellules exprimant que HA-TSG101, puits 1, en présence de NCp7-eGFP, puits 2, ou de NCp7ZF1-eGFP, puits 3, ou de NCp7ZF2-eGFP puitst 4 ou NCp7ZF1ZF2-eGFP, puits 5.

Figure 8 :

 8 Figure 8 : Pourcentage de FRET et de la population d'eGFP-TSG101 interagissant avec Gag vue par FLIM.Les cellules sont transfectées avec les différentes constructions et analysées par FLIM en utilisant une loi biexponentielle permettant de calculer le temps de vie (converti en FRET en utilisant le temps de vie du contrôle eGFP seul, histogramme A) et la population associée à ce FRET (histogramme B).

  (NC)(D'Souza and Summers 2005; Lever 2007; Muriaux and Darlix 2010; Rein 2010; Lu, Heng et al. 2011; Rein, Datta et al. 2011). First it has been shown that GagNC displays a strong preference for viral RNA and RNAs containing the Ψ site

Table 1 : List of plasmids used in my thesis: Plasmid Resistance gene Tag Promot eur Provenance

 1

	eGFP	Kanamycine		CMV
					Dr. D.Ott (National Cancer
	Gag-TC	Ampicilline	/	CMV	Institute, Frederick, Maryland,
					USA)
	Gag-eGFP	Ampicilline	C-terminal (MA)	CMV	Dr.V.Goldshmidt
	Gag-mCherry	Ampicilline	C-terminal (MA)	CMV	Dr.V.Goldshmidt
	Gag-∆ZF1-TC	Ampicilline	/	CMV	Dr.V.Goldshmidt
	Gag-∆ZF2-TC	Ampicilline	/	CMV	Dr.V.Goldshmidt
	Gag-∆ZF1∆ ZF2-TC	Ampicilline	/	CMV	Salah EL Meshri
	Gag-∆NCp7-TC	Ampicilline	/	CMV	Salah EL Meshri
	Gag-∆p6-TC	Ampicilline	/	CMV	Salah EL Meshri
	Gag-L6i7-TC	Ampicilline	/	CMV	Salah EL Meshri
	Gag-eGFP-∆ZF1	Ampicilline	C-terminal (MA)	CMV	Salah EL Meshri
	Gag-eGFP-∆ZF2	Ampicilline	C-terminal (MA)	CMV	Salah EL Meshri
	Gag-eGFP-∆ZF1∆ ZF2	Ampicilline	C-terminal (MA)	CMV	Salah EL Meshri
	Gag-eGFP-∆NCp7	Ampicilline	C-terminal (MA)	CMV	Salah EL Meshri
	Gag-eGFP-∆p6	Ampicilline	C-terminal (MA)	CMV	Salah EL Meshri

1.3. Primary and secondary antibodies

Table 2 : List of different primary antibodies used

 2

	Name	Species	Provider	Reference	Fluorophore or enzyme	Mono Polyclonal	/	Application / dilution
	Anti-p24 Gag	Mouse	NIH	6521	/	Monoclonal		WB 1/10000
	Anti-p24 Gag	Rabbit	abcam	32352	/	Monoclonal		WB 1/10000
	Anti-HA	Rabbit	abcam		/	Polyclonal		IP 4µg/mg WB 1/4000
	Anti-eGFP	Mouse	proteintech 66002-1-Ig	/	Monoclonal		WB 1/4000
	Anti-TSG101	Rabbit	abcam	Ab30871	/	Polyclonal		WB 1/250
	Anti-HA	Mouse	abcam		/	Monoclonal		WB 1/10000
	Anti-GAPDH	Mouse	millipore	MAB374	/	Monoclonal		WB 1/5000
	Anti-mCherry	Rabbit	abcam		/	Polyclonal		WB 1/1000
	Anti-RPS7	souris	abcam	57637	/	Monoclonal		WB 1/500
	Anti-RPS14	lapin	abcam	50390	/	Polyclonal		WB 1/400
	Anti-Flag	souris	sigma	F1804	/	Monoclonal		WB 1/4000

Table 3 : List of different secondary antibodies used

 3

	Name	Species	Provider	Reference	Fluorophore or enzyme	Mono Polyclonal	/
	Anti-mouse HRP		Promega	W402B	HRP	WB 1/10000
	Anti-rabbit HRP		Promega	W401B	HRP	WB 1/10000
	Anti-Rabbit	Goat	invitrogen		Alexa fluor 488 IF 1/2000
	Anti-Mouse	Goat	invitrogen	A110111	Alexa fluor 568 IF 1/2000
	protein A		invitrogen	101023	HRP	WB 1/15000
	1.4. Oligonucleotides

Synthetic oligodeoxyribonucleotide (ODN) were purchased from Sigma-Aldrich. The sequences of these oligonucleotide primers used in my thesis are described in the table below:

Table 4 : The oligonucleotides used for mutagenesis are phosphorylated at the 5 ' end.

 4

  2 in DMEM solution, Glutamax (Dulbecco's

	Modification of Eagle's Medium) developed by Harry Eagle in 1959. This medium contains
	amino acids (L-Threonine, L Glutamine), salts (potassium chloride and magnesium sulfate),
	vitamins (folic acid, nicotinamide, and riboflavin), glucose and organic supplements such as
	pyruvate. It is supplemented with fetal calf serum or 10% FCS and 5 ml mixture of antibiotics,
	names as S/P, like penicillin (100 IU/ml) and streptomycin (100 IU/ml). Serum would be used as
	cell growth factors which they need to proliferate and antibiotics are used to prevent bacterial
	contamination.
	The first step was to remove the culture medium. Then the cells were washed with 10 mL of 1X
	PBS (Phosphate Buffered Saline 10X: KH 2 PO 4 1.44g/L NaCl 9 g/L, NaHPO 4 7.95 g/L, pH 7.4).

Table 5 :

 5

			Number of adherent cells to deposit	Maximum amount of DNA (μg)	Volume DNA (μL)	Volume NaCl (μL)	Volume jetPEI® (μL)
		tube 1		/	/	46	4
	12 wells plate		80 000 -100 000
		tube 1'		2	X	50-X	/
	6-well plates or μ-Dish 35mm Ibidi	tube 1 tube 1'	200 000 -300 000	/ 3	/ Y	94 100-Y	6 /

Table 5 :

 5 Number of HeLa cells to be deposited in the wells and conditions of use jetPEI® used for transfection of HeLa cells

Table 6 : Mutagenesis Reaction 96

 6

  Les étapes tardives du cycle de réplication du VIH correspondent à l'assemblage des protéines structurales Gag, le bourgeonnement de la particule et sa maturation. Beaucoup de travaux ont été menés sur ces différents aspects. Cependant, le rôle joué par les différents partenaires à savoir les protéines virales et cellulaires et les acides nucléiques restent à préciser d'un point de vue mécanistique et dans le cadre d'une recherche de molécules douées d'activités anti virale. La primo-infection est suivie d'une phase asymptomatique ; il s'agit de la séroconversion. Dans cette phase de latence, il n'y a pas de symptômes, le système immunitaire est efficace, il y a un équilibre entre la multiplication et la réaction immunitaire qui se maintient un certain temps. La charge virale augmente progressivement tandis que le nombre de lymphocytes T CD4+ diminue lentement.

	PhD Student: EL MESHRI Salah Edin
	PhD Director: DE ROCQUIGNY Hugues (hderocquigny@unistra.fr)
	UMR 7213 -Laboratoire de biophotonique et pharmacologie
	Préambule :
	I -Introduction :
	I-1-Le VIH et le SIDA
	Le virus de l'immunodéficience humaine (VIH) qui est responsable du SIDA est un lentivirus
	enveloppé qui appartient à la famille des retroviridae. Depuis sa découverte en 1983 on estime
	que 30-35 million de personnes de part le monde sont décédées et qu'environ autant de
	personnes sont de nos jours infectées. En France, le nombre de personne concerné est d'environ
	150 000 pour un nombre de décès annuel de 6000 cas. Sa transmission s'effectue essentiellement
	par les fluides biologiques lors de rapports sexuels non protégés, lors de l'allaitement ou de
	transfusion sanguine.
	L'infection se passe en plusieurs étapes (Figure 1). Schématiquement, 15 à 30 jours suivant la
	contamination, le virus se réplique activement. Cette phase se traduit dans 75% des cas par
	quelques symptômes comme fièvre, ganglions, éruptions cutanées. Les cellules infectées par le
	VIH correspondent aux lymphocytes T CD4+ helper (ou auxilaire), en particulier les cellules T CD4+
	mémoires ainsi que les cellules dendritiques, les macrophages… qui sont impliquées dans
	l'initiation de la réponse T auxiliaire et de l'amplification des diverses fonctions du système

immunitaire. La cytotoxicité du virus (détournement de la machinerie cellulaire à son profit) ou de certaines de ces protéines conduit a une déplétion rapide du système immunitaire.

  On voit apparaître de graves symptômes tels que l'amaigrissement rapide, fièvres longues et intenses, diarrhées persistantes, ganglions importants, infections pulmonaires inhabituelles. A ce stade le déficit immunitaire est sévère, la mort survient par des maladies opportunistes et non par le virus du SIDA lui-même. On observe un brusque infléchissement de la pente de déplétion des cellules CD4+ 6 à 18 mois avant le survenu du stade SIDA et un déclin rapide jusqu'à la disparition complète des lymphocytes T CD4+ au stade SIDA. La plupart des patients infectés par le VIH, dans les pays occidentaux, reçoivent un traitement de type HAART (highly active antiretroviral therapy), soit une association de trois médicaments provenant d'au moins deux classes d'antirétroviraux. Ce traitement vise à obtenir une suppression maximale et durable du VIH, d'une charge virale indécelable. est de restaurer un

	nombre de lymphocytes T CD4+ . Plusieurs classes d'antirétroviraux existent à savoir des anti RT
	(eux-mêmes divisés en deux familles de type nucléosidique ou non nucléosidique), des anti
	protéases et plus récemment des anti fusions (qui ne sont plus préscrits), des anti CCR5 et des
	anti intégrase.
	Le génome code également six autres protéines dites protéines auxiliaires ou régulatrices : Nef,
	Rev, Vif, Vpr, Vpu et Tat
	Au cours de ma thèse, je me suis focalisé essentiellement sur la protéine Gag. Elle est
	composée de différents domaines, matrice (MA), capside (CA), nucléocapside (NCp7) et de p6.
	• La matrice (MA) est une protéine de 132 acides aminés et présente un poids moléculaire
	de 17 kDa. Cette protéine possède plusieurs signaux de ciblage (signal d'adressage à la

I-2-Structure du virus : Le VIH-l forment des particules sphériques d'un diamètre de 80 à 120 nm et sont produits par bourgeonnement à la surface des cellules infectées. Ces particules sont formées d'une enveloppe externe et d'une nucléocapside dense et excentrée en forme de trapèze ou de barreau à l'intérieur de la particule virale. L'enveloppe est constituée d'une bicouche lipidique d'origine cellulaire et de deux glycoprotéines virales formant les spicules: la gp120 et la gp41. Elle est tapissée d'une matrice (MA) sur sa face intérieure. Le coeur du virus est formé par la protéine de CA entourant la nucléocapside (NCp7) qui renferme le génome viral constitué de deux molécules d'ARN de haut poids moléculaire ainsi que les enzymes nécessaires à sa réplication: la transcriptase inverse, l'endonucléase ou intégrase et la protéase.

Figure 2 : Structure du virus HIV-1 Le génome du VIH-1 est constitué de deux brins d'ARN non épissés identiques, d'environ 9.2 kb, maintenus ensemble par des interactions non covalentes. Le site majeur d'interaction des ARN, nommé « Dimer Linkage Structure » (DLS), a été visualisé par microscopie électronique au niveau des extrémités 5' (1,2). Ces molécules d'ARN sont issues d'une transcription par l'ARN polymérase II cellulaire et sont donc toutes deux coiffées à leur extrémité 5' et polyadénylées à leur extrémité 3'. Le génome viral comporte 3 régions codantes principales, correspondant aux séquences des polyprotéines Gag, GagPol et Env. Elles codent respectivement pour les protéines de structure, les enzymes virales et les protéines d'enveloppe. membrane, signal de localisation nucléaire, signal d'export nucléaire) régulés par des

Results and Discussion

Summary in French

Figure 5 : Rôle de la NC dans l'assemblage de Gag Distribution du FRET et de la population de Gag engagée dans l'interaction à 12h (a et c) et à 24h (e et g). Les moyennes de ces distributions sont représentées sous forme de 'boites à moustaches'. Triangle noir et rond noir correspondent à Gag et GagNC alors que les couleurs magenta, vert et bleue correspondent aux mutants GagZF1, GagZF2 et GagZF1ZF2.

Acknowledgments

First, I would like to thank Prof. Yves MELY, director of laboratory Biophotonics and Pharmacology UMR CNRS 7213 of the Faculty of Pharmacy of Strasbourg for giving me the opportunity to do my thesis in his laboratory. I would like to thank all members of the Jury Dr. Gilles MIRAMBEAU, Dr.Serge BOUAZIZ and Dr. Marc RUFF for having kindly agreed to evaluate my work. Acknowledgements I would also like to thank the Cultural Affairs in Libyan embassy, ministry of high education, National Agency for Scientific Research and Biotechnology Research Center for the financial support of my thesis. I wish to warmly thank the whole team of "biophotonics and pharmacology" and more specifically: Dr. Hala EL MEKDAD, Sarwat ZGHEIB, Nedal TAHA, Emilie CHRIST, Hassan KARNIB, Manuel PIRES, Waseem ASHRAF, Redouane BOUCHAALA, Liliyana ZAAYTER , Dr. Avisek GOSH, Rajhans SHARMA, Noémie, Marianna, Ievgen, Lesia and Vasyl. To my parents who have always supported me to go through with my ambitions, who trusted me and gave me confidence. A special thanks to my wife who was next to me in all difficult stages of my thesis, for her encouragement and support every day. To my lovely children Elmutasmbellah, Kinan and Miral. A warm thanks to my brothers and my sisters for their love and support. Acknowledgments: SIDACTION, ANRS, THINPAD, INSERM, CNRS, UDS, NIH Reagents, KS for fruitful discussion, Ott et Barbara for plasmid

Influence of the nucleocapsid region of the HIV-1 Gag polyprotein precursor on the recruitment and trafficking of the Tumor susceptibility gene 101.

En conclusion, la polymérisation de Gag est initiée dans le cytoplasme, bien avant de s'accumuler au niveau de la membrane plasmique. Dans nos études nous montrons que le domaine NC de Gag est important lors des étapes d'assemblage et de trafique de Gag dans la cellule. Les défauts observés avec la protéine GagNC sont probablement dus à un défaut de reconnaissance de l'ARN, interaction essentiellement médiée par le domaine NC et indispensable à la polymérisation de Gag dans le cytoplasme. Une étude est maintenant en cours pour suivre cette interaction entre les ARN cellulaires et la protéine Gag en utilisant une technique développée au laboratoire (71).

III-2-Rôle potentiel de la NC dans l'interaction Gag-TSG101

Les travaux ci dessus ont aussi confirmé (non montré) que l'expression de GagNC se traduisait par une nette diminution de la production des particules virales (50 ,72 ,73).

Cependant, cette construction possède les deux domaines tardifs PTAP and LYPX n L de la p6 qui interagissent avec la protéine TSG permettant l'export des particules virales. Nous avons donc pris comme hypothèse que le domaine GagNC pouvait aussi être impliqué dans le bourgeonnement du virus. Dans un premier temps nous avons effectué des expériences de co précipitation entre la protéine TSG101 et différentes constructions de Gag (figure 6). Nous montrons ainsi que la protéine Gagp6 est peu efficacement précipitée par TSG101 (comparer figure 6C, puits 3 avec Gag et puits 7 pour Gagp6) en accord avec les données de la littérature montrant l'importance du domaine PTAP de p6 dans le recrutement de la TSG101 (28,30,74 ,75). Cependant, le signal restant dans le puits 7 sous entend qu'une autre partie de Gag puisse être importante dans cette interaction. A la lumière du défaut de bourgeonnement observé avec les mutants de Gag, nous avons poursuivi ces expériences avec les constructions de Gag délétées de la NC ou simultanément des deux motifs dactyles. and LYPXnL late domains to recruit the cellular machinery necessary for viral budding, PLoS pathogens 5, e1000339 (34) Popov, S., Popova, E., Inoue, M., and Gottlinger, H. G. (2008) Cimarelli, A., Sandin, S., Hoglund, S., and Luban, J. (2000) Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA, J Virol 74,[3046][3047][3048][3049][3050][3051][3052][3053][3054][3055][3056][3057]