COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) infection is a long-term disease which required a long-life treatment. Besides the standard highly active retroviral therapy (HAART) regiment, HIV-1 gene therapy is one of a promising alternative strategy which give rise to hope for better HIV-1 treatments. In addition, protein-based therapeutics represent another promising approach which show the high impact results in curing various types of diseases. Nowadays, it has already become a significant part of the current medical treatments. In this study, we focused on αRep proteins, the nonimmunoglobulin scaffold proteins which were designed to target nucleocapsid (NC) domain of HIV-1 Pr55Gag polyprotein precursor and investigated their possible roles as intracellular therapeutic agents. Phage display technology was used for the specific isolation of αRep against a critical C-terminal region of the HIV-1 Pr55Gag (CA21-SP1-NC) from a large and diverse αRep phage library. Several strong αRep binders were thus isolated, but only two binders, referred to as αRep4E3 and αRep9A8, were further characterized. The αRep4E3 contains 7 internal repeat motifs (32 kDa), whereas αRep9A8 has 6 repeat motifs (28 kDa). Both αRep proteins were expressed at high level in a conventional bacterial expression system, and were recovered at high yields in soluble form. Their potential antiviral activity was investigated using different cell systems. The baculovirus system was used for determining the antiviral effects on virus-like particle (VLP) production of both αRep scaffold molecules. Sf9 cells stably expressing N-myristoylated αRep4E3 and αRep9A8 fused to the GFP reporter protein at their C-termini were infected with a recombinant baculovirus carrying the gene encoding the HIV-1 Pr55Gag polyprotein (AcMNPV gag ), and the antiviral effects were evaluated by quantitative and qualitative methods. The results showed that N-myristoylated αRep4E3 and αRep9A8 qualitatively altered the particle formation and were coencapsidated with Pr55Gag into the VLPs released into the culture supernatant.

The different patterns of particle morphology were observed by electron microscopy.

The αRep4E3-expressing cells mainly produce aberrant VPLs, while αRep9A8 expression induces the accumulation of Pr55Gag at the plasma membrane of AcMNPV gag -infected Sf9 cells. In the human T cell line system, αRep4E3 and αRep9A8 proteins were expressed intracellularly by transducing SupT1 cell with SIN CGW lentiviral vector carrying the αRep genes fused to the EGFP coding sequence at their 3'-end. These stable SupT1 cell lines were then challenged with HIV-1NL4-3. The results indicated that both αRep4E3 and αRep9A8 displayed antiviral effects, which occurred at the late steps of the HIV-1 life cycle, with no effect on proviral DNA integration.

Reduction and delay in the viral progeny production were found in infected SupT1 expressing αRep4E3-EGFP and αRep9A8-EGFP. Difference in the antiviral mechanism was observed between these two αRep proteins: αRep4E3-EGFP mainly interferes with the packaging of the viral genomic RNA, while αRep9A8-EGFP interferes with the proteolytic processing of Pr55Gag polyprotein, and performs as a protease inhibitor to prevent the HIV-1 protease cleavage which is required for the production of newly infectious mature virions. Interestingly, SupT1 expressing αRep9A8-EGFP is able to survive to chronical HIV-1 infection up to 38 days after infection, with a low level of noninfectious HIV-1 particle production. Taken together, our results suggested that αRep, a new type of scaffold protein, could serve as a promising alternative antiviral agent which would influence the future strategies and candidates of antiviral molecules to be used in anti-HIV-1 cell therapy.

ACKNOWLEDGEMENTS

This thesis is the story which has been arisen for several years in my Ph.D. study.

I would say that it is such a great and wonderful memories. However, I will never forget experiencing many important lessons, hard examinations, late night experiments and the stressful I have when I wrote this thesis. During my study program, I have had a very great honor to work with a large number of amazing people. It would not have been possible to finish this thesis without their warm help, support, guidance, and friendship.

It also would not have been possible to finish this thesis without financial support throughout my education. I would like to take this opportunity to express that this thesis is the result of many experiences I have encountered from dozens of remarkable individuals who I wish to acknowledge.

First and foremost I wish to express my deepest gratitude and sincere appreciation to my thesis supervisor, Prof. Dr. Chatchai Tayapiwatana, for giving me a great opportunity to be a Ph.D. student under his supervision. It has been a great honor to be his student, not only because of his tremendous academic support but also because he is one of the great teachers and researchers I have ever met in my life. His guidance helped me in all the time of studying and writing of this thesis. He has always made himself available to clarify my doubts despite his busy schedules. I could not have imagined having a better advisor and mentor for my Ph.D. study. He is the type of person called "polymath" which means "a man can do all things if he would like to". I really wish that someday I could be as enthusiastic and energetic as Prof. Chatchai and to someday be able to command an audience as well as he can.

I am extremely grateful to express my gratitude and appreciation to Dr. Saw-See Hong and Prof. Pierre Boulanger, my thesis advisors from UMR754, UCBL, and my parents in Lyon for their support, guidance, and taking care of me. They are the beginning of alpha repeat proteins project, the beginning of this thesis work. Dr. Saw-See is the one that changed my perspective on life. I see things more in a positive way after I know her.

I have learned to be more kind, sweet, and be a positive thinking person than I was.

Prof. Pierre is a rare type of man I know, I mean it in a good way. He is like a big mobile library with a large storage capacity. He always makes me surprise that how can one man packs all that knowledge inside the brain, not only about sciences but also many others; history, archeology, architecture, and politic, and everything. I must thank you for all of your kind, supportive, encouragement, insightful comments and hard questions during thesis writing and manuscript preparation. Finally, I would like to thank you for taking care of me like a family, without you two, living in France for two years could not be this happy. 2. This thesis contains a part of the study that used the newly developed method for investigating the ratio of mature per immature virus particle in the sample. This new technique was developed based on the competitive ELISA combined with using the unique anti-MA antibody which recognizes the cleaved C-terminal of MA when processed by HIV-1 protease enzyme.

CHAPTER 1 INTRODUCTION

Long-term treatment of Human Immunodeficiency Virus 1 (HIV-1) infection can be readily achieved by combinations of antiretroviral agents, usually referred to as Highly Active Antiretroviral Therapy (HAART). The combination of antiviral drugs can result in the suppression of plasma viremia to less than the limit of quantification (< 50 copies/ml) with consequential improvement in the level of CD4+ T cell counts.

This was which associated with the resolution of established opportunistic infections although they cannot eradicate the viruses due to latent viral reservoirs. In addition, prolonged treatment tends to result in the occurrence of multi-drug resistant mutants, drug-drug interaction and drug toxic effects. All these drawbacks justify the exploration of alternative therapeutic approaches such as gene therapy and cellular therapy in order to enhance the anti-HIV-1 response (1-8).

In the past 30 years we have witnessed the feasibility of gene and protein therapies for the treatment of diverse human diseases. These approaches apply the powerful genetic engineering which is applicable to the treatment of both genetic and acquired maladies ranging from blood diseases to the treatment of infectious diseases such as HIV infection.

Antibody therapy is one of the successful strategy in which a molecule of antibody or antibody-derived protein scaffold is used as the therapeutic agents. Although it is one of the major commercial success of the biotechnology industry to date, antibody molecules have their limitations [START_REF] Simeon | In vitro-engineered non-antibody protein therapeutics[END_REF][START_REF] Ewert | Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering[END_REF].

In the past decade the development of protein engineering techniques had made it possible to generate novel repeat scaffold protein binders called non-immunoglobulin scaffolds.

The absence of disulfide bond in these scaffold molecules is the key factor which makes them suitable for intracellular applications, particularly inside the cytosolic compartment in which the proper folding of bioactive proteins should not be influenced by the reducing conditions. In addition, unlike antibodies, scaffold proteins have less complexity in the production processes. They can be expressed in bacterial cell systems with high production levels, high solubility and high stability. For these reasons, a number of scaffold molecules are now in the preclinical and clinical development stage. DARPins, the designed ankyrin repeat proteins which are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. The human immunodeficiency virus is a member of the genus Lentivirus, belonging to the Retroviridae family. When the viruses enter the body, they attack the immune system by specifically infecting the CD4+T cells, which help the immune system fight against infections. If improperly treated or left untreated, HIV reduces the number of CD4+T cells in the body, making the infected person susceptible to microbial infections or infection-related cancers. These opportunistic infections or cancers take advantage of the deficient immune system provoked by the viruses, called acquire immune deficiency syndrome (AIDS), the last stage of the HIV infection. There are two types of HIV, HIV-1 is the cause of the majority of HIV infections globally due to the high rate of viral production, while HIV-2, mainly present in Western Africa, is less pathogenic and shows a much lower rate of disease development [START_REF] Gilbert | Comparison of HIV-1 and HIV-2 infectivity from a prospective cohort study in Senegal[END_REF][START_REF] Popper | Lower human immunodeficiency virus (HIV) type 2 viral load reflects the difference in pathogenicity of HIV-1 and HIV-2[END_REF]. In this study the literature review will be focused mainly on the HIV-1.

1.) HIV-1 life cycle

HIV-1 begins its life cycle when the mature viruses infect their target cells This figure has been obtained from the site: http://www.nature.com/nrmicro/journal/v10/n4/full/nrmicro2747.html

2.) HIV-1 structural proteins

The proteins and gene functions of HIV-1 have been the subject of extensive research since the discovery of the virus in 1980s [START_REF] Barre-Sinoussi | Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)[END_REF][START_REF] Barre-Sinoussi | Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)[END_REF]. The formation of HIV-1 particles is driven by the viral structural protein, Gag polyprotein precursor (Pr55Gag) [START_REF] Gheysen | Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells[END_REF]. The specific cleavage of Pr55Gag precursor by HIV-1 protease enzyme (PR) generates four major structural components of the mature virions including matrix (MA), capsid (CA), nucleocapsid (NC), and p6 protein and two spacer peptides (SP) which include SP1 and SP2 as shown in (Figure 1.2) [START_REF] Liang | Characterization of a putative alpha-helix across the capsid-SP1 boundary that is critical for the multimerization of human immunodeficiency virus type 1 gag[END_REF][START_REF] Henderson | Gag precursors of HIV and SIV are cleaved into six proteins found in the mature virions[END_REF]. Within the mature virions, MA domain is layered underneath membrane envelop meanwhile the shell of the core structure made of CA proteins. Inside the viral core, NC is tightly associated with the 2 copies of viral genomic RNA [START_REF] Gelderblom | Assembly and morphology of HIV: potential effect of structure on viral function[END_REF][START_REF] Wilk | Organization of immature human immunodeficiency virus type 1[END_REF]. To be assemble into the new virions, the process begins at the inner leaflet of plasma membrane of

3.) Matrix (MA)

The MA protein is the domain of Pr55Gag which is located on the N-terminal side of the Gag precursor. The MA plays an important role in the trafficking of Pr55Gag to the plasma membrane. This process is mediated by its Nmyristic signal, added to the N-terminus of Pr55Gag by cytoplasmic Nmyristoyl-transferase co-acting with the Gag protein translational machinery.

The membrane binding function of Gag is mediated by the insertion of myristoyl group of the myristic acid into the lipid bilayer particularly, the acidic phospholipid, PI(4,5)P2. Moreover, the highly conserved patch of basic residues of MA promotes the Gag membrane interaction. Apart from the membrane targeting function, MA is required for the incorporation of envelope (Env) glycoproteins, gp120 and gp41 into the virus particles. In addition, several studies showed that MA contains a RNA binding site which overlaps the PI(4,5)P2 binding domain. The interaction of MA with nucleic acid has been suggested to enhance the selectivity of MA for PI(4,5)P2-rich cellular membrane and prevent premature intracellular assembly [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF][START_REF] Sundquist | HIV-1 assembly, budding, and maturation[END_REF][START_REF] Ono | Relationships between plasma membrane microdomains and HIV-1 assembly[END_REF][START_REF] Ganser-Pornillos | The structural biology of HIV assembly[END_REF][START_REF] Hill | Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly[END_REF][START_REF] Gottlinger | Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1[END_REF][START_REF] Ono | Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane[END_REF][START_REF] Chukkapalli | Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient gag membrane binding[END_REF].

4.) Capsid (CA)

The CA domain of Gag is critical for the viral particle assembly. In mature viral particles, CA forms the conical shape viral core observed in mature virions [START_REF] Gelderblom | Assembly and morphology of HIV: potential effect of structure on viral function[END_REF]. The viral CA can be divided into two domains which have different functions in the particle morphogenesis. The N-terminal domain of CA (CANTD) comprises of two third of CA (Figure 1.2 bottom). CANTD is necessary for the mature core formation, but lacks the function necessary for the immature Gag polymerization process [START_REF] Borsetti | The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly[END_REF]. The C-terminal domain of CA (CACTD) is critical for both core formation and Gag oligomerization/polymerization in the particle assembly process [START_REF] Ono | Relationships between plasma membrane microdomains and HIV-1 assembly[END_REF][START_REF] Mateu | The capsid protein of human immunodeficiency virus: intersubunit interactions during virus assembly[END_REF][START_REF] Mcdermott | Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteinespecific reagents[END_REF][START_REF] Maldonado | New insights into retroviral Gag-Gag and Gag-membrane interactions[END_REF].

It has been reported that mutations in CACTD severely impede viral particle formation and Gag-Gag interaction (37).

5.) Nucleocapsid (NC)

The NC region of Gag, which lies downstream of the CA domain, has been shown to be important for the viral genomic RNA packaging and promoting Gag-Gag interactions [START_REF] Ganser-Pornillos | The structural biology of HIV assembly[END_REF]. The NC domain contains two conserved CCHC zinc finger motifs coordinated with two zinc ions linked by a basic RAPRKKG sequence, and both N-terminal and C-terminal domain are flanked by flexible sequences [START_REF] Didierlaurent | The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation[END_REF]. There is evidence suggesting that this domain is absolutely essential for the virus replication. The function of NC involves its interaction with RNA stem loops, a hundred nucleotide-long portion of the viral RNA which locates at the 5'-end of the untranslated region (UTR) of the viral RNA, and on the 5'-side of the gag initiation codon. This region contains the packaging signal, the so called Psi (ψ), which is required for the efficient encapsidation of the unspliced viral genome. Inside the mature virus particle, NC is found within the CA core where several molecules of NC coat the two copies of viral genetic material [START_REF] Didierlaurent | The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation[END_REF]. Moreover, NC harbors another domain called interacting domain (I domain), which consists of basic residues mediating Gag-Gag interaction. Most of the studies review that primarily 'tethers' Gag molecules together via RNA bridges, then additional NC-NC interactions are underwent. Then NC-RNA tether complexes promote the increasing of Gag concentration at the assembly site, resulting in the enhancement of CA-CA interaction. [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF][START_REF] Ganser-Pornillos | The structural biology of HIV assembly[END_REF][START_REF] Maldonado | New insights into retroviral Gag-Gag and Gag-membrane interactions[END_REF][START_REF] Didierlaurent | The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation[END_REF][START_REF] Jalaguier | Efficient production of HIV-1 virus-like particles from a mammalian expression vector requires the N-terminal capsid domain[END_REF]. More interestingly recent studies show that NC also plays a key role for chaperoning nucleic acid during viral DNA synthesis by HIV-1 reverse transcriptase enzyme and prevents nonspecific self-priming that induced by TAR stem-loop structure at 3'end of minus strand ssDNA (40, 41).

6.) Spacer peptide 1 and spacer peptide 2 (SP1 and SP2)

During the viral maturation step, HIV-1 Gag precursor protein is cleaved by viral PR to obtain the major mature proteins which include MA, CA, NC, and p6 domain. Beside these major domains, there are two spacer peptides, SP1

and SP2, which act as a linker between CA-NC and NC-p6, respectively, and are released after the PR cleavage [START_REF] Liang | Characterization of a putative alpha-helix across the capsid-SP1 boundary that is critical for the multimerization of human immunodeficiency virus type 1 gag[END_REF][START_REF] Krausslich | The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity[END_REF]. The SP1 region has been shown to be essential for the assembly of viral like particles (VLP). There are two functional properties of the SP1 domain which have been characterized.

Firstly, the importance of the ~6 first residues of SP1 which are involved in the particle formation. Minor changes in the first 4 residues drastically destroy the correct particle assembly in mammalian cells [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] Datta | On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch[END_REF]. This study suggested that the proper assembly probably requires the basic residues at the N-terminus of SP1. Secondly, several reports indicate that SP1 promotes the strength of Gag-Gag interaction. In summary, SP1 is required for the immature particle assembly and in regulating the formation of the mature HIV capsid essential for virus infectivity [START_REF] Gottlinger | Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1[END_REF][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] Datta | On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch[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] Pettit | The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions[END_REF].

SP2 is the domain that separates NC from the p6 domain. Unlike SP1, the function of SP2 in virus morphogenesis and infectivity are not currently well characterized. However, there is a study reporting that the blockage of both cleavage sites between NC and p6 did not affect processing of other Gag cleavage sites upstream, but caused a severe reduction of viral infectivity, close to background levels [START_REF] Coren | Mutational analysis of the C-terminal gag cleavage sites in human immunodeficiency virus type 1[END_REF][START_REF] Kafaie | Role of capsid sequence and immature nucleocapsid proteins p9 and p15 in Human Immunodeficiency Virus type 1 genomic RNA dimerization[END_REF][START_REF] Muller | HIV-1 Gag processing intermediates trans-dominantly interfere with HIV-1 infectivity[END_REF]. In addition, another study provides evidence that the release of virus particles does not require the presence or the proteolytic processing of SP2. The viral infectivity is almost abolished when both cleavage sites (the "slow" cleavage site between NC and SP2, and the "fast" cleavage site between SP2 and p6) are mutated, and severely reduced when the fast cleavage site SP2-p6 is altered. This correlates with an increased proportion of irregular core structures, although processing of CA is not affected. Mutation of the slow cleavage site NC-SP2, or deletion of most of SP2, had only minor effects on infectivity and did not induce major alterations in mature core morphology. These results suggest that the processing kinetics leading to the separation of NC and p6 is essential for successful maturation of the virions, while SP2 itself is dispensable (49).

7.) p6 protein

While MA, CA, and NC are the common proteins found in all retroviral Gag precursors, the p6 domain is a specific feature of HIV-1 and other primate lentiviruses. In the full-length Gag molecule, p6 is located at the C-terminus and separated from the NC by the short spacer peptide SP2. The p6 domain is encoded by a sequence which directs translational frameshifting into the overlapping pol open reading frame. Thus, p6 is the only Gag domain which is absent from the Gag-Pol polyprotein [START_REF] Gottlinger | Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release[END_REF]. It has been shown that, within the viral particle, p6 is the major phosphoprotein in the mature form, but the function of this phosphorylation remains unclear. By contrast, a major function of p6 has been well characterized: it consists of the release of assembled viruses from the cell surface and/or from each other, which is highly dependent upon p6. In the sequence of p6, the major determinant of this function is the conserved P(T/S)APP motif near the N-terminus of the p6 domain. This region is now known as the late assembly (L) domain.

Moreover, the L domain can induce the recruitment of cellular factors to the site of particle assembly (51).

8.) HIV-1 genome

The full HIV-1 genome is encoded in one long strand of RNA. In the free virus particle, there are two copies of single-stranded positive-sense RNA Between the two LTR regions, the viral genome contains the genes encoding the three structural polyproteins Gag, Pol, and Env, the genes encoding four accessory proteins Vpu, Vif, Vpr, and Nef, and the genes for the two regulatory proteins Rev and Tat [START_REF] Mcburney | Viral sequence diversity: challenges for AIDS vaccine designs[END_REF][START_REF] Nkeze | Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe[END_REF]. with acute myeloid leukemia (AML). His physician therefore, arranged for him to receive a hematopoietic stem cell transplant from a donor with the "delta 32" mutation on the CCR5 receptor results in a mutated CCR5 protein [START_REF] Hutter | Longterm control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF][START_REF] Novembre | The geographic spread of the CCR5 Delta32 HIV-resistance allele[END_REF]. Those individuals who are homozygous for the CCR5 mutation are resistant to HIV and rarely progress to AIDS. The majority of HIV cannot enter a human cell without a functional CCR5 gene. An exception to this is only a small minority of viruses that use alternate receptors, such as CXCR4 or CCR2 [START_REF] Alkhatib | The biology of CCR5 and CXCR4. Current opinion in HIV and[END_REF]. To prevent fusion of the virus with the host membrane, enfuvirtide (INN) is one another example of inhibitor in this class. Enfuvirtide is a peptide drug that must be injected into the body and acts by interacting with the N-terminal heptad repeat of gp41 of HIV to inactivate the hetero sixhelix bundle formation, therefore preventing infection of host cells [START_REF] Lalezari | A phase II clinical study of the long-term safety and antiviral activity of enfuvirtide-based antiretroviral therapy[END_REF].

11.) Nucleoside reverse transcriptase inhibitors

HIV-1 is an RNA virus, to be able to integrate its genome into the host chromosome, it must be reverse transcribed into the DNA. Since the conversion of RNA to DNA is not done in the mammalian cell, it is performed by a viral protein which makes it a selective target for inhibition. For

13.) Integrase strand transfer inhibitors

The

15.) Assembly and maturation inhibitors

The assembly of Gag proteins into immature viral particles followed by proteolytic disassembly of the Gag shell to mature capsids are pivotal steps for the generation of infectious HIV-1 [START_REF] Braun | HIV-1 capsid assembly inhibitor (CAI) peptide: structural preferences and delivery into human embryonic lung cells and lymphocytes[END_REF]. The identification of active peptides as candidates for interfering negatively with the viral assembly step is a promising strategy [START_REF] Sundquist | HIV-1 assembly, budding, and maturation[END_REF]. The HIV-1 CA protein plays essential roles in viral replication and as such represents an attractive new therapeutic target.

In 2005, a 12-mer peptide termed capsid assembly inhibitor (CAI), identified by phage display, was reported to disrupt both immature-and mature-like particles in vitro by targeting the C-terminal domain (CTD) of HIV-1 CA [START_REF] Sticht | A peptide inhibitor of HIV-1 assembly in vitro[END_REF]. However, the CAI peptide itself fails to inhibit HIV-1 assembly in cell culture due to its inability to penetrate cells. A structure-based rational design approach known as hydrocarbon stapling has then been developed to stabilize the alpha-helical structure of CAI and convert it to a cell penetrating peptide.

The resulting molecules, named NYAD-1 and NYAD-13, efficiently disrupt HIV-1 assembly in cell cultures. However, because they have a relative low affinity for CA, these peptides are unlikely to progress to the clinic [START_REF] Bukrinskaya | HIV-1 assembly and maturation[END_REF].

More recently, a third small-molecule binding site on CA was identified via Therefore, the alternative treatments to combat HIV are required, one promising alternative approach is gene therapy, i.e. the use of therapeutic individual surfaces to specifically bind to their target. These structures have special repeat at the N-terminus and C-terminus of the protein with a hydrophilic surface which function as a 'cap' to shield the hydrophobic core from the solvent. This modular structure introduces numerous possibilities for binding a diverse set of ligands in a specific manner. Repeat proteins follow rules derived from biophysical consideration, such as a large interaction surface necessary for tight binding. Such a binding interface should be rigid, in order not to lose conformational entropy upon binding, which would otherwise decrease the overall achievable binding free energy. The surface should be modular so that they can be individually exchangeable or modifiable by affinity maturation. These proteins do not require disulfide bonds, which allow their expression in bacterial cytoplasm [START_REF] Tamaskovic | Designed ankyrin repeat proteins (DARPins) from research to therapy[END_REF][START_REF] Boersma | DARPins and other repeat protein scaffolds: advances in engineering and applications[END_REF].

Several well-known repeat protein families have been studied and characterized, to define the set of rules for the creation of the new artificial protein types. For examples, the successful design of full consensus leucine-rich repeats domain (LRRs), ankyrin (AR), and tetratricopeptide (TPR) repeats has been reported. More recently, the design of consensus armadillo (ARM) and HEAT repeats proteins has been reported as well (87)

1.) αRep proteins

Recently, the new artificial repeat protein family named αRep have been reported. Their design was based on thermostable HEAT-like repeats, in which HEAT stands for Huntingtin, Elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase TOR1 (HEAT). These proteins were shown to be well-expressed, well-folded, and extremely stable [START_REF] Urvoas | Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (alphaRep) based on thermostable HEAT-like repeats[END_REF][START_REF] Guellouz | Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep)[END_REF].

HEAT repeats firstly identified in the eukaryotes and later were found in the prokaryotes as common motifs. The biological functions of this protein are very diverse, but most of them are mainly involved in protein-protein interactions. Although HEAT repeats are very common, using them for artificial scaffold proteins library is not very widespread, maybe because of the size and irregularity of their repeats. These proteins fold as the α-helix Moreover, accommodation of pIII to the large proteins and peptides cause little loss of functionality related to the ability of the phage infectivity to the bacteria. By comparison, the pVIII protein, although much more abundant (2,700 copies per phage) can display only short peptides (6-8 amino acids), due to geometric constrains during capsid assembly of the phage particle.

pairs
Phage-display techniques have two major advantages for many research field as well as the antiviral research. They can be used for the rapid and high-throughput screening of large numbers of different molecules (up to 10 13 ) in a small volume (a few microliters). For example, the privilege of selecting a phage with a frequency of 1/10 8 in the original library has been reported. In addition, phage display defines a physical link between genotype and phenotype, because the gene encoding the protein (or peptide) displayed at the surface is directly sequenced and isolated from the phage DNA. Furthermore, directly used as expression vectors obtain from some of phagemid vector, to produce the protein or peptide of interest. Finally, using bacteriophages in a display strategy has several advantages for example (i) the genome of phage is small size, this simplifying the cloning steps; (ii) phage particles are stable, which can store at 4°C; (iii) the high efficiency of phage infection, allow the possibility to obtain the large amount of material, compatible with the screening of large libraries; (iv) phage-display libraries can be periodically regenerated, while chemical libraries consumed over time.

Nevertheless, the phage-display technology also has several drawbacks that have to be taken into account. For example, the clones can be toxic for the bacterial cells leading to the reduction of bacterial growth, or may decrease the infectivity of phage and/or phage particle secretion. Some clones are unlikely to be selected will therefore disappear rapidly from the pool library, regardless of the selection process. In addition, the expression level of certain peptide sequences in E.coli is variable (depending on the codon usage), introducing another potential bias into the bacteria may affect selection process. Finally, successive amplifications of the library may rapidly decrease the diversity of the library in order to the clones with a growth advantage are likely to increase significantly within the population, at the expenses of the other clones. In another unfortunate case, the selected peptides or proteins, once synthesized as soluble form, those molecules may not retain their optimal binding activity outside of the microenvironment of the phage and/or in the complex environment such as cell or animal. The phage-display of combinatorial peptide libraries has already been used successfully several times over the last 20 years, in the discovery of inhibitory peptides active against viruses [START_REF] Hong | Protein ligands of the human adenovirus type 2 outer capsid identified by biopanning of a phage-displayed peptide library on separate domains of wild-type and mutant penton capsomers[END_REF]. The first viruses to be targeted were hepatitis B virus (HBV) and HIV, and both these two

Baculovirus

In the early 1980s, the first-publication about the use of the baculovirus vector for the expression of a foreign gene led to a wave of great interest for recombinant protein production using baculovirus in insect cell systems. The Baculoviridae is a large family of viruses which harboring double stranded, circular DNA genomes.

The natural hosts for these viruses are arthropods, and mainly insects. However, This virus infects the gut epithelial cells and virus replication takes place. BV is then produced and buds from the infected gut cells. At this point, the virus spreads throughout the tissues of its new host. Although the polyhedrin protein is essential for survival of the virus in nature, it is dispensable for virus survival and propagation in tissue culture cells. The first one is, the BV, once budded from the site of primary infection, is free to infect neighboring cells by the pathway which has been described above. Secondly, OCV is released from occlusion bodies after an infected food source is ingested by a new host [START_REF] Murphy | Overview of the baculovirus expression system. Current protocols in protein science[END_REF]. The figure has been obtained from Murphy CI et al., 2001.

most

Lentivirus vectors

Lentiviral vectors are among the most popular and useful viral vectors in many research fields and clinical gene therapies. The advantages of lentivirus that make it attractive as a tool for gene-delivery vectors, are the followings: (i) it can generate a stable vector integration into the host genome; (ii) it has a large genetic capacity of transgene insertion; (iii) a broad range of tissue tropisms; (iv) and the ability to transduce both dividing and non-dividing cells [START_REF] Sakuma | Lentiviral vectors: basic to translational[END_REF][START_REF] Mccarron | Challenges of up-scaling lentivirus production and processing[END_REF]. In the early era of lentiviral vectors, genetically modified HIV was constructed as gene delivery vehicle. The first prototype was the HIV based-vector with the components of viral vector separated into two plasmids, a plasmid encoding HIV-1 proviral DNA with a deletion in the env gene and a second plasmid that encodes only env gene [START_REF] Helseth | Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants[END_REF][START_REF] Page | Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity[END_REF].

Since the trans-complementation of Env protein is in a separated plasmid, the viral vector is produced by a single-round infection. In this HIV viral vector the transgene was designed to be inserted into the nef or env gene and its expression is controlled by the 5'LTR. The HIV-1 based vector was also constructed to have the essential cis-acting elements for genome packaging, reverse transcription and integration, and expression of genome transcripts, i.e. the ψ signal, the LTRs and RRE, respectively. In addition, the foreign gene was controlled by an external promoter such as the immediate-early cytomegalovirus (CMV) promotor. Since HIV-1 Env required the CD4 molecule at the cell surface as specific receptor, the range of cell types susceptible to this gene transfer system was broadened by introducing the vesicular stomatitis virus envelope glycoprotein G (VSV-G) into the viral vector production system. VSV-G binds to the ubiquitous membrane component phosphatidylserine, and this makes these VSV-G pseudotyped vectors able to transduce a broad range of cell types, not only mammalian cell but also non-mammalian cells such as fish cell (100). The VSV-G pseudotyping offers additional advantages to the vectors: for instance, VSV-G is substantially more stable than retroviral or lentiviral envelopes; consequently the VSV-G pseudotyped vectors can be concentrated by ultracentrifugation to obtain high-titer vector stocks.

Lentiviral vectors are derived from the highly pathogenic virus HIV-1. Therefore, using this technology, the most important issue that have to be considered is the biological safety, and more specifically the possibility of generating replication competent lentiviruses (RCLs). The first generation of lentivirus vectors was made to reduce this risk. Hence, replication-deficient recombinant HIV-1 vectors are produced from three separate elements (i) a packaging construct; (ii) a plasmid encoding the Env glycoprotein; and (iii) a transfer vector which contains the gene of interest (Figure 1.7 (A)) The packaging construct harbors the genes which code for HIV Gag, Pol and regulatory/accessory proteins, under the control of a strong mammalian promoter. The second plasmid is the env-encoding plasmid, the mostly used is VSV-G, to provide the advantageous broad range of host cells. These two plasmids were specifically engineered to lack any packaging signal or LTRs to avoid their transmission into vector particles and to reduce the production of RCL in vector preparations. The plasmid vector which carries the gene of interest contains all the cis-acting elements required for packaging, reverse transcription, integration and expression of the transgene (ψ signal, LTRs and RRE), without the expression of any HIV protein. However, since the trans-activator Tat is not encoded together with the transgene in the transfer vector, the activity of the 5'-LTR promoter is minimal. Therefore, for the protein production from the transgene, an internal promoter specific of the transduced cells is used to control the transgene expression.

To further increase the biosafety, second-generation vectors have been developed by modifying the accessory genes in the system (Figure 1.7 (B)). HIV-1 contains four accessory protein which are Vif, Vpu, Vpr, and Nef, which are necessary for the propagation of the virus and its virulence in primary cell infection. Although these accessory genes are important for HIV as a pathogen, they were deleted in second-generation lentiviral vectors (101). Therefore, these second-generation vectors include only four of the nine HIV genes: gag, pol, rev and tat.

To be able to integrate into the host chromosome by functional integrase, the cassette of the transfer vector must be flanked with LTRs on both 5'-and 3'-ends.

E. coli strains

XL-1 Blue (Stratagene, La Jolla, CA) was used as a host for the αRep phage library generation and amplification. M15[pREP4] bacterial cells (Qiagen, Hilden, Germany)

were used for production of soluble 6xHis-tagged αRep proteins. BL-21 was uses as a host for the preparation of GST-tagged recombinant truncated Gag domains.

Plasmids and vectors

The pQE-31 expression vector (Qiagen, Hilden, Germany) was used for the production of 6xHis-tagged recombinant αRep proteins in M15[pREP4] bacterial cells. pIB/V5-His (Invitrogen, Carlsbad, CA) was used for the construction of insect Sf9 cell lines stably expressing αRep scaffold molecules under the control of OplE2 promoter for their constitutive expression. Blasticidin (Gibco, Grand Island, NY) was used as a selection agent to obtain a stable polyclonal population or stable clonally cell lines. The pCEP4 vector (Invitrogen, Carlsbad, CA) was used as an episomal mammalian expression vector of αRep proteins, with the cytomegalovirus (CMV) immediate early enhancer/promoter for high level expression of transgene. pCEP4 vector carries the hygromycin B resistance gene for stable selection in transfected cells. CGW, a third-generation of lentiviral vector, was used as the backbone vector to transfer genes encoding for αRep proteins into Human embryonic kidney (HEK) 293T cells and SupT1 cells. The pGEX-4T-1 vector (Stratagene, La Jolla, CA) was used in this study to produce the high level expression of GST-tagged recombinant truncated Gag domains. pNL4-3 was used to produce the HIV-1 virus.

Cells

The Sf9 cell line was derived from pupal ovarian tissue of the fall armyworm Spodoptera frugiperda (Invitrogen, Calsbad, CA). This cell line is highly susceptible for the infection of Autographa california nuclear polyhedrosis virus (AcNPV) and can be used with the baculovirus expression vectors. Sf9 cells were used to generate the stable cell lines expressing αRep proteins fused to GFP protein. Cells were propagated and maintained in non-humidified environment at 27 °C in Grace's insect medium (Gibco, Grand Island, NY) supplemented with 10 mM L-glutamine, (Gibco, Grand Island, NY), 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY), penicillin (100 Units/ml), and streptomycin (100 μg/ml).

Human embryonic kidney (HEK293T) cell obtained from the American Type Culture Collection (Manassas, VA) and human cervical cancer cell line (HeLa), a generous gift from Prof. Dr. Andre Lieber (University of Washington, Seattle, WA, USA) were maintained in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, Grand Island, NY)

supplemented with 10% FBS (HyClone, Cramlington, UK), 2 mM L-glutamine, penicillin (100 Units/ml) and streptomycin (100 μg/ml).

Human T cells lymphoblastic lymphoma (SupT1) cell were obtained from the ATCC (Manassas, VA) were grown in RPMI-1640 medium containing penicillin (100 U/ml), streptomycin (100 μg/ml), 2 mM L-glutamine, and 10% FBS (all cell lines were maintained in a humidified 37°C incubator containing 5% CO2.

Construction of αRep library

The αRep library was previously constructed by the polymerization of synthetic microgenes which correspond to the repeats in the phage display or periplasmic expression vector. The intermediate vector construct called acceptor vector (pHDiEx-acc) was constructed. This vector was used for low level expression of αRep proteins fused to the M13 g3p truncated protein in phage display experiments. Since this construction had also a T7 promoter and a suppressible stop codon between the αRep protein and g3p coding sequences, it could also be used for periplasmic expression of non-fused αRep protein in non supE strains of E. coli expressing T7 polymerase. For cytoplasmic expression, the sequence encoding soluble αRep proteins were inserted into the pQE-31 expression vector, using M15[pREP4] strain for protein expression. Bacterial cells, XL-1 Blue were used as a host cells for generation of the library and propagation of phages displaying the artificial αRep proteins. The αRep library was kindly provided by Professor Philippe Minard, University Paris-Sud, Orsay.

Screening of αRep phage library on the viral targets

The viral target used, was the GST-CA21-SP1-NC polyprotein derived from HIV-1

GagPr55 including the 21 amino acids of C-terminal domain of CA fused to SP1 and NC.

This protein was used for the screening of phage-displayed αRep library. The αRep clones that bind to immobilized target GST-CA21-SP1-NC were isolated. Phage clones were amplified and three rounds of selection/elution step were repeated. The elution of GST-CA21-SP1-NC bound phages were performed using acidic buffer for first two rounds, followed by specific ligands elution using excess of GST-CA21-SP1-NC as competitors in the third round. Individual phage particles were then prepared in small scale using the 96-wells format. Briefly, random colonies from each round of selection

Expression and purification of αRep proteins M15[pREP4] bacterial cells harboring pQE-31-αRep recombinant plasmids were

grown in 500 ml Luria-Bertani (LB) broth supplemented with ampicillin 100 μg/ml, kanamycin 25 μg/ml, and 1% (w/v) glucose, at 37°C with shaking up to OD600 of 0.8 was reached. Protein expression was induced by addition of 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), and maintained in culture for 4 hours at 30°C

Western immunoblotting

To determine the αRep proteins expression, bacterial cells were pelleted, and washed once with PBS then resuspended in PBS containing a cocktail of protease inhibitor (Roche Diagnostics, Germany). The bacterial cells were physically lysed by sonication. After clarifying the cell lysate by centrifugation, the concentration of protein was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Equal amounts of total protein were separated by 12% SDS-PAGE and transferred to PVDF membrane (GE healthcare, UK). After blocking with 5% skim milk in PBS, the PVDF membrane was incubated with mouse monoclonal anti-6xHis (Applied Biological Materials (ABM) Inc, Richmond, Canada), and probed with HRP-conjugated goat antimouse antibody (KPL, Gaithersburg, MD). The immunoreactive proteins were visualized by color development. A, αRep4E3-EGFP and B, αRep9A8-EGFP.

Construction of

Production of VSV-G-pseudotyped lentiviral vectors

VSV-G-pseudotyped lentiviral vector particles were produced in HEK293T cells, using four separate plasmids and transfected into the cells by calcium phosphate co-transfection method as the schematic showed in Figure 2.4. Briefly, co-transfection of CGW transfer vector 10 μg/dish with the packaging construct pRSV-Rev 2.5 μg/dish, pMDLg/pRRE 6.5 μg/dish, and pMD.2G 3.5 μg/dish into 70-80% confluent HEK293T cells in 10-cm dish was performed. At 16 hours post-transfection fresh C-DMEM containing 5% FBS was replaced and cells were further incubated. The particles of lentiviral vector were harvested from the culture supernatant at 24 and 48 hours post-transfection. They were filtered through sterile syringe filters with a 0.45 μm pore size (Millex-HA filter unit;

Merck Millipore, Hessen, Germany). The higher-titer of viral vector stocks was prepared by 20% sucrose cushion ultracentrifugation at 100,000 x g, in 4˚C, for 2 hours and 20 Note: the titer must be calculated from a sample corresponding to a vector dilution when fluorescence positivity of cells ranges between 5%-25%, in order not to underestimate the titer when multiple transduction events per cell have occurred.

Construction of the stable

HIV-1 stock

Replication-competent X4 tropic virus HIV-1NL4-3 virus was produced by transiently transfect the pNL4-3 plasmid into the HEK293T cells. Briefly, monolayers of HEK293T cells were seeded into the 10-cm dish at 4×10 6 cells to obtain the monolayer of the producer cells follow by transfected with 5 μg of the pNL4-3 plasmid using transIT-X2 transfection reagent (Mirus Bio, Madison, WI) according to the manufacturer's protocol.

The cells were allowed to grow in the culture medium for 48 hours. HIV-1 virus particles in culture supernatant were then harvested and filtrated through the sterile syringe filters with a 0.45 μm pore size. HIV-1 stock was aliquoted and stocked at -80°C. The virus titer was determined using conventional p24 antigen ELISA, using the Genscreen ULTRA HIV Ag-Ab assay (Bio-Rad, Marnes-la-Coquette, France). The viral load was determined using the COBAS AMPLICOR HIV-1 Monitor test (Roche Molecular Systems, Branchburg, NJ).

Evaluation of antiviral activity of αRep proteins in SupT1 stable cell lines

The level of intracellular antiviral protection conferred by myristoylated αRep4E3EGFP, non-myristoylated αRep4E3EGFP, myristoylated αRep9A8EGFP, and nonmyristoylated αRep9A8EGFP were determined by challenging these cell lines with the X4-tropic virus, HIV-1NL4-3. Control SupT1 cells and SupT1 cells stably expressing αRep proteins, were cultured in the fresh growth medium for at least 4 weeks before HIV-1 challenging. To perform the infection assay, spinoculation of target cells were performed with 1 MOI of HIV-1 for 16 hours. The cells were next washed three times using serumfree medium, and resuspended in fresh growth medium. At 3-day intervals, cells were split (1:2) to maintain the density of the cells. The yields of HIV-1 replication was monitored in the culture supernatants using p24 antigen ELISA (Bio-Rad, Marnes-la-Coquette, France) and viral load assay (COBAS AMPLICOR HIV-1 Monitor test, version 1.5; Roche Molecular Systems, Branchburg, NJ). The cell pellets were kept for the determination of the proviral integration level. The cell viability was performed in parallel by the trypan blue dye exclusion staining assay.

ELISA based HIV-1 Pr55Gag maturation assay

Microtiter plates were coated with 100 μl of 5 μg/ml of mouse anti-MA HB-8975 monoclonal antibody diluted in coating buffer 1M NaHCO3 pH 8. There is competition when the culture supernatant contains the mature form (free cleaved MA) of Gag protein or if HIV PR is able to specifically cleave at MA-CA junction and free C-terminal of MA is exposed. The same epitope present as a biotinylated synthetic p17MA C-terminal peptide, is recognized by an anti-MA HB-8975 monoclonal antibody.

The reaction were then reviewed by streptavidin conjugated HPR. The TMB micro well peroxidase substrate 100 μl were added after 1 hour incubation time followed by stopped reaction with 1 N HCl and measured the optical density OD at 450 nm.

Generation of HIV-1 target proteins

Our viral targets consisted of various GST-fused polyproteins derived from the HIV-1

GagPr55 precursor (LAI isolate). GST-CA21SP1-NC included the 21 amino acids of the C-terminal domain of the capsid protein (CA), fused to the SP1 linker and the whole nucleocapsid (NC) domain, encompassing its two zinc fingers (ZF) until residue F433.

A series of C-truncated mutants of GST-CA21SP1-NC were also constructed (Figure 2.5).

The DNA fragments coding for each construct were rescued by PCR amplification from pGEX-GST-CA21SP1-NC, using specific oligonucleotide primers. The amplification products were purified by GeneJET PCR Purification kit, and a first step gene cloning was performed using InsTAclone PCR cloning kit (ThermoFisher Scientific, Waltham, MA). XL-1 Blue competent cells were transformed by the ligation products, and plated on LB agar containing ampicillin (100 μg/ml). Colonies were picked for PCR amplification, restriction enzyme analysis using BamHI and XhoI, and DNA sequencing.

The DNA fragments of each construct were isolated from the pTZ57R/T vector and transferred to pGEX-GST expressing vector using the same restriction sites, BamHI and XhoI. Three pGEX-GST-gag plasmids were thus obtained: (i) pGEX-GST-CA21SP1-NC;

(ii) pGEX-GST-CA21SP1-NCΔZF2; and (iii) pGEX-GST-CA21SP1. BL21 cells were used for recombinant Gag protein production. BL-21 harboring each of the recombinant pGEX-GST-gag plasmids were cultured in 200 ml LB broth containing 100 μg/ml ampicillin and 1% (w/v) glucose, at 37°C with shaking. When OD600 reached 0.8, protein expression was induced by addition of 0.1 mM IPTG, and maintained in culture at 30°C overnight with shaking. Cells were pelleted by centrifugation at 1,200 x g for 30 min at 4°C. Cell pellets were resuspended in PBS containing a cocktail of protease inhibitors (Roche Diagnostics GmbH), then lysed by sonication. Soluble proteins were purified by affinity chromatography on glutathione-agarose gel (ThermoFisher Scientific), following the protocol of the manufacturer, and analyzed by SDS-PAGE and Western blotting.

Protein pull-down assays

The E. coli BL21 cells were transformed with plasmids pGEX-4T1 harboring the genes encoding the GST-CA21-SP1-NC, GST-CA21-SP1-NC∆Zf2, and GST-CA21-SP1

proteins, and bacterial cells cultured in Terrific broth (TR) medium supplemented with ampicillin (100 μg/mL), and glucose (1%; w/v) at 37°C with shaking. When the OD600 of the cultures reached 0.8, protein expression was induced by addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside, and the culture was further maintained at 30°C for 16 hours with shaking. Bacterial cells were then pelleted by centrifugation at 1,200 x g at 4°C for 30 min, resuspended in PBS containing a cocktail of protease inhibitors (Roche Diagnostics GmbH), and sonicated. After clarification by centrifugation (15,000 x g at 4°C, for 30 min), aliquots of supernatants containing GST-CA21-SP1-NC, GST-CA21-SP1-NC∆Zf2, or GST-CA21-SP1 protein were incubated with purified αRep4E3 or αRep9A8 protein (300 μg) in PBS-0.01% Tween-20 (1 ml total volume), for 2 hours at RT with shaking. Aliquots (20 μl) of glutathione (GSH)-coated agarose beads (Pierce Glutathione Agarose; Thermo Scientific, Waltham, MA) were then added to the reaction mixture, and further incubated for 1 hour at RT with shaking. Beads were centrifuged at low speed, washed 4 times with 0.05% Tween-20 in PBS, and resuspended and heat-denatured in SDS-PAGE loading buffer.

CHAPTER 3 RESULTS

A new scaffold protein library and the CA21-SP1-NC domain of the Gag polyprotein precursor as target

In this PhD project, we used a new repeat-protein library called alpha-Repeat (αRep)

which was constructed and provided by the lab of Prof. Philippe Minard (CNRS Paris-Orsay). In brief, the structures and sequences of a new type of artificial repeat protein, HEAT-like repeat was described. From the known structure of a thermostable HEAT repeat subfamily, the consensus sequences of the HEAT repeat protein were identified and devised to become N-Cap and C-Cap. The protein library was then assayed the biophysical properties. The results obtained suggested that in this library, the proteins differ by the number of internal repeat motifs and the local amino acid residues of each repeat in the variable positions. The αRep proteins have advantages over the ankyrin repeat proteins in particular, their thermostability (Tm > 70°C) and other properties discussed in Chapter 1. Since the aim of our study was to isolate molecules which would negatively interfere with the morphogenetic process of the virus particle, we screened the αRep library for binders against the CA21-SP1-NC domain of the Gag precursor. The SP1 linker is a short, 14-residue peptide acting as a hinge between the capsid (CA) and the nucleocapsid (NC) domains of the Pr55Gag polyprotein precursor and has been shown to be essential for the correct oligomerization of Pr55Gag and the assembly of membraneenvelope virus particles but mainly involved in the virus maturation and infectiveness.

The NC domain is essential for assembly, encapsidation of the viral genome, and acts as nucleic acid chaperone [START_REF] Sundquist | HIV-1 assembly, budding, and maturation[END_REF][START_REF] Didierlaurent | The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation[END_REF].

The SP1 domain is framed by two crucial protease cleavage sites, and this region is essential for the immature particle assembly and in regulating the formation of the mature HIV-1 capsid essential for virus infectivity [START_REF] Datta | On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch[END_REF][START_REF] Pettit | The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions[END_REF]. For all the reasons mentioned above,

SP1

Selection of CA21-SP1-NC binders from a library of phage-displayed αRep proteins

The phage display is the method to express binder peptides or proteins of interest at the surface of phage particles. The peptides or proteins which are interactors of a given target can be identified by affinity selection, a technique called "bio-panning" (109). The phage display system allows production of protein or peptide libraries with a high diversity of randomized ligands, making it a very versatile and high-throughput screening method for the identification of potential binding molecules specific for different types of target molecules of interest. In this study, the αRep scaffold phage library used for our screening against the CA21-SP1-NC domain of Pr55Gag has been constructed and described in

Biochemical and biophysical characterization of CA21-SP1-NC binders

To characterize the six αRep protein clones obtained from phage-ELISA, the αRep genes were sub-cloned into the plasmid expression vector pQE-31, and verified by DNA code for the amino acids alphabet). However, they differed both in the number of their repeats (7 and 6 repeat motifs were found for αRep4E3 and αRep9A8, respectively) and in the nature of residues found at the variable positions (presented in red alphabet).

Therefore, each internal repeat of αRep contains five random variable amino acids which serves as the keys binding residues. In summary, the difference in repeat numbers and in amino acids occupying the variable positions in αRep4E3 and αRep9A8 explained their difference in binding activity towards the viral target.

After protein production and purification, a single band of αRep protein was observed in SDS-PAGE analysis, migrating at 32 kDa for αRep4E3 and at 28 kDa for αRep9A8

(Figure 3.4 B & D).
The high intensity of both αRep protein bands over the background of bacterial proteins suggested that αRep4E3 and αRep9A8 were expressed at high yields, and in a highly soluble form, in the bacterial expression system. Moreover, our results

indicated that αRep4E3 and αRep9A8 were able to retain their target binding function in vitro. In the next step, these two αRep candidates were further tested for their function inside the cell, using the insect cell expression system.

Effects of αRep4E3 and αRep9A8 on HIV-1 virus-like particle assembly

Since αRep4E3 and αRep9A8 showed a significant binding activity towards the CA21-SP1-NC domain of HIV-1 Pr55Gag in vitro, we next explored their effect on the assembly of HIV-1 virus-like particle (VLP) in vivo, in a heterogenous cell expression system. The first experiment was performed using the recombinant baculovirus-infected insect cells system.

Construction of Sf9-derived cell lines stably expressing αRep proteins

The genes of the αRep4E3 and αRep9A8 were designed and fused to the green VLPs were isolated by ultracentrifugation through a 30%-50% sucrose-D2O gradient.

Each fraction was pelleted through isopropanol, lysed and assayed for VLP-incorporated luciferase activity [START_REF] Gonzalez | Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay[END_REF]. A, Schematic diagram illustrating the strategy to evaluate the possible inhibitory effects of αReps on HIV-1 VLP assembly, using luciferase assay. B, Western blot analysis of extracellular, pelletable Gag polyprotein using anti-Gag The electron microscopic analysis showed that these VLPs could adopt different shapes such as oval or irregular particles, the latter consisting of twin-, triple-, or multi-particles budding inside the same membrane envelope. These "multiparticle buds" were more particularly visible in sections of Sf/(Myr+)αRep4E3 cells (Figure 3.7 B-F), although irregular particles were also frequently observed in sections of Sf/(Myr+)αRep9A8 cells (Figure 3.8 (B-D)). However, the aberrant assembly of VLPs in Sf/(Myr+)αRep9A8-GFP cells showed an additional and specific pattern, consisting of the accumulation of Gag protein at the inner leaflet of the plasma membrane, an effect which was not observed with αRep4E3 (Figure 3.8 E-G). The results of this EM observation showed that both αRep4E3 and αRep9A8 negatively interfered with the HIV-1 Gag assembly process, but in two different ways, and thus that αRep4E3 and αRep9A8 exhibited two distinct phenotypes. The level of HIV-1 CAp24 antigen was determined in the culture supernatants collected at different days (D) after challenge (D3, D7, D14, and D21 pi), and the cell morphology monitored by phase-contrast microscopy (Figure 3.12). The results

showed that the CAp24 antigen could only be detected at D14 pi in samples from SupT1/αRep4E3-EGFP and SupT1/αRep9A8-EGFP cells, with an increased level at D21. By contrast, in control, non-transduced SupT1 cells and SupT1αRep/9C2-EGFP cells, the CAp24 antigen was observed earlier, at D7 pi, with a decrease at D21. The CAp24 level in both SupT1/αRep4E3-EGFP and SupT1/αRep9A8-EGFP samples was therefore ~ 10-fold lower at D14 pi, compared to its level in samples from control, non-transduced SupT1 cells and SupT1/αRep9C2-EGFP cells.

However, this negative effect was partial and apparently transient, and the maximum virus particle production delayed, from D14 pi (as in the control samples) to D21 pi (Figure 3.12 B). This suggested that the two αRep proteins exerted some inhibitory function against HIV-1, by altering the kinetics of viral progeny production.

The morphology of HIV-1-infected cells, as observed under the light microscope, revealed that HIV-1 infection induced syncytium formation and cell lysis at earlier times in both types of control cells, non-transduced SupT1 cells and SupT1/αRep9C2-EGFP cells, compared to SupT1/αRep4E3 and SupT1/αRep9A8.

Of note, the observed decrease of CAp24 levels in control samples at D21 pi compared to their augmentation in SupT1/αRep4E3 and SupT1/αRep9A8 samples, was likely due to the cell death, which drastically reduced the number of HIV-1infected, CAp24-producer cells, as shown in Figure 3.12 C. The addition of new fresh culture medium to the wells every 3 day to maintain the culture alive diluted the extracellular CAp24 antigen.

To ensure that our lentiviral vector-modified SupT1 cell lines were able to maintain Our results indicated that the antiviral effects mediated by αRep4E3 and αRep9A8 took place at a late step of the HIV-1 replication cycle, after the integration step.

Both αRep proteins were able to significantly, but transiently, reduce the viral production by SupT1 cells, as assessed by the CAp24 assay and genome copy titration. However, significant differences were observed in the biological properties of the two αReps: (i) αRep4E3 seemed to be more efficient than αRep9A8 in the reduction of extracellular viral genomes, and thus more detrimental

Mapping of the αRep binding sites on the viral Gag target

The respective position of the αRep4E3 and αRep9A8 binding sites on the CA21-SP1-NC

CHAPTER 4 DISCUSSION

HIV-1 infection is a long-term disease which requires a long-term treatment. Although

HAART is an effective strategy for the treatment of HIV-1 infected patients and the control of plasma viremia under detectable level. Nevertheless, there are limitations that emerged over time when it is becomes a long-life treatment. For example, prolonged treatment can resulting in the occurrence of multi-drug resistant mutants, drug-drug interaction and drug toxicity (1)(2)(3)[START_REF] Spreen | Long-acting injectable antiretrovirals for HIV treatment and prevention[END_REF](5)[START_REF] Tang | HIV-1 antiretroviral resistance: scientific principles and clinical applications[END_REF](7)[START_REF] Arenas | Genetic Consequences of Antiviral Therapy on HIV-1[END_REF]111). For these reasons, alternative treatment have been proposed. HIV-1 gene therapy was introduced as a promising alternative treatment for infected patients [START_REF] Arts | HIV-1 antiretroviral drug therapy[END_REF][START_REF] Scherer | Progress and prospects: RNA-based therapies for treatment of HIV infection[END_REF][START_REF] Bennett | Gene therapy strategies for HIV/AIDS: preclinical modeling in humanized mice[END_REF]112). These include the identification or design of genes coding for intracellular factors or interactors with antiviral activity, the genetic manipulation of hematopoietic progenitor stem cells, and the inactivation of proviral DNA in situ by using zinc-finger nucleases (ZFNs)(113), transcription activator-like effector nucleases (TALENS) (114), or the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system (115, 116). Protein therapeutics is also another promising alternative which has become an integral and significant part of current medical treatments (117). The well-known examples of this next generation medicine include engineered antibodies, bi-or multi-specific proteins, antibody mimic novel scaffold molecules, single chain variable fragment [START_REF] Simeon | In vitro-engineered non-antibody protein therapeutics[END_REF][START_REF] Gebauer | Engineered protein scaffolds as next-generation antibody therapeutics[END_REF]118,119). Nevertheless, antibody therapy has their own limitations therefore, the paradigm shifted to the non-immunoglobulin binding proteins. The advantages of protein scaffolds overpower the limitations of the antibody-based therapies make them promising candidates for clinical applications. Their independence of disulfide bonds concurs them the ability to express in reducing environment such as the cytoplasmic compartment of cells.

Moreover, scaffold proteins are highly soluble and exhibit thermal stability. They are smaller than an antibody molecule which is an advantage for tissue penetration and accumulation. In addition, the production or synthesis of scaffold proteins are less Due to the target CA21-SP1-NC is covered a part of C-terminal domain of CA, SP1 region, and NC domain. The evidence showed that CACTD is critical for both core formation and Gag oligomerization/polymerization in the particle assembly process [START_REF] Ono | Relationships between plasma membrane microdomains and HIV-1 assembly[END_REF][START_REF] Maldonado | New insights into retroviral Gag-Gag and Gag-membrane interactions[END_REF]122).

Moreover, SP1 region is required for the immature particle assembly by promoting the strength of Gag-Gag interaction and regulating the formation of the mature HIV-1 CA which is essential for virus infectivity [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] 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]. The studies also reviewed that SP1 region especially first six residues are necessary for the normal-regular particle assembly. NC domain not only is involved in the viral genome packaging, the formation of NC-RNA tether complexes promote the increasing of Gag concentration at the assembly site, resulting in enhanced CA-CA interaction (125). Furthermore, CA-NC contains two PR cleavage sites, interact with αRep proteins which may interfere with Gag-PR interaction or accessibility of an enzyme. All these suggested that the aberrant particle formations may be the cause of the reasons mention above [START_REF] Gheysen | Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells[END_REF][START_REF] Ganser-Pornillos | The structural biology of HIV assembly[END_REF]125). Subtle differences observed in the αRep9A8-GFP expressing Sf9 cells is the fringe pattern, the accumulation of Gag protein at the plasma membrane without ability to generate the new particles. This maybe because of their interaction with αRep9A8-GFP resulting in the failure of Gag polymerization. However, we are able to observed Gag on the cell membrane because

Gag itself are able to address to the plasma membrane via the myristic signal at its N-terminus. With this experiment, the results showed the promising negative effect of αRep4E3 and αRep9A8 in terms of the quality of VLPs production but not effect on the amount of VLPs. Based on the baculovirus expression system used in this study is which known as the most powerful and versatile protein expression system, it could be that the level of Gag protein expression are saturated, αRep proteins are therefore not enough to compete with these high level of Gag expression. In addition, AcMNPV gag can re-infect cells overtime to produce more Gag protein. Meanwhile, αRep proteins are constitutively expressed from absolute number of genome copies integrated in the Sf9 cells host chromosome.

It was very interesting that aberrant VLPs were released by the αRep4E3 and αRep9A8 expressing Sf9 cells. One hypothesis was that the abnormal particle morphology was caused by the incorporation of αRep proteins into the VLPs and bud out together. To test this hypothesis, sucrose gradient ultracentrifugation were performed, and the results confirmed that both αRep4E3-GFP and αRep9A8-GFP were encapsidated inside the infection. We therefore, further analyzed the maturation and infectivity of the HIV-1 viruses released from αRep4E3 and αRep9A8 expressing SupT1 cells. ELISA-based maturation assay revealed that at the equal amount of CAp24, αRep4E3 is likely not to be able to interfere or display a negative effect on the virus maturation as its percentage maturation was nearly 80% which is in the same level of SupT1 and SupT1 αRep9C2 irrelevant control. It could be presumed that these virus particles are able to undergo maturation and become mature form. However, the viral infectivity assay of HIV-1 viruses released from αRep4E3 contradictory. We could expect that the mature form of particles could have a high viral infectivity. But the results showed that their infectivity were significantly lower than the controls, suggesting that αRep4E3 has no effect on viral maturation but interfered at the late phase of HIV-1 life cycle by another pathway.

According to the viral load assay, it was shown that the genome copies detected in the culture supernatant of SupT1/αRep4E3-EGFP at D14 pi was the lowest level. Therefore, the mode of action of αRep4E3 may involve the genome packaging. Another plausible hypothesis could be that αRep4E3 may interfere the late stage reverse transcription regulation of NC (126). Binding of αRep4E3 to the NC domain may promote the reverse transcription at the viral assembly step, following packing the viral DNA and egress.

Interestingly, αRep9A8 expressing SupT1 cells produced immature virus particles higher than other SupT1 stable cell lines and SupT1 cell control. This antiviral effect was confirmed by subjecting viruses released from αRep9A8 expressing SupT1 cells to the viral infectivity assay. The results indicated that they were able to re-infect the Jurkat-GFP at very low levels compared to the SupT1 and SupT1/αRep9C2-EGFP irrelevant control, suggesting that αRep9A8 has antiviral effects against viral maturation and viral infectivity. The data obtained from the percentage viability and total cell number strongly supported the antiviral effect of αRep9A8. After SupT1/αRep9A8-EGFP cells were infected with a high amount of viruses and most of infected cells in the culture well died, resulting in few HIV-1 producer cells. Subsequently, SupT1 cells expressing αRep9A8

were able to resist by releasing immature virus particles every time that new virions are produced, resulting in no new infections. Therefore, SupT1/αRep9A8-EGFP were able to grow and the cell viability as well as number increased as shown in D38 pi. However, background level of virus particle production were found, but more interestingly, the viral infectivity assays showed that the extracellular HIV-1 particles released by SupT1/αRep9A8-EGFP at D38 pi were noninfectious.

All together we can conclude that both αRep4E3 and αRep9A8 displayed antiviral effects against HIV-1 replication by reducing and delayed the virus production as same as level of HIV-1 genome in culture supernatant. In addition, they were able to interfere with the infectivity of the virus. However, the molecular mechanism of action of these two αRep proteins are different as the schematic showed in The mapping of the αRep4E3 and αRep9A8 binding sites on the viral target provided some clues to explain their functional differences as antivirals. The most plausible hypothesis for the inhibitory effect of αRep9A8 protein on virus maturation would be that αRep9A8 would block the PR-mediated maturation cleavage of the Gag precursor indirectly, by steric hindrance, and not via a direct masking of the SP1 sites. Meanwhile, αRep4E3 would interact with amino acid residues belonging to the two adjacent zinc fingers of NC which resulting in the reducing of genomic RNA packaging. Mixed and stored at -20 ºC.

Luciferase assay substrate

Tris

Faculté

  Au cours de notre programme de thèse, nous avons isolé et caractérisé des molécules protéiques à activité antivirale intracellulaire dirigée contre le VIH-1. Ces protéines, appelées αRep, ont été obtenues par criblage d'une banque de protéines artificielles de nouvelle génération, construites de façon combinatoire à partir de protéines naturelles constituées de motifs alpha-hélicoidaux répétés. La cible virale (ou "appât") utilisée pour ce criblage est une région de la polyprotéine Gag du VIH-1 identifiée comme une cible privilégiée de nouvelles thérapeutiques antivirales, car essentielle à l'assemblage viral, l'empaquetage du génome viral et le clivage de maturation de Gag aboutissant à la formation de virions infectieux. Deux molécules d'αRep à forte affinité pour la cible virale, l'αRep4E3 (32 kDa; 7 motifs répétés) et l'αRep9A8 (28 kDa; 6 motifs répétés) ont ainsi été isolées, clonées et caractérisées. L'étude de l'activité anti-VIH intracellulaire de ces αRep a été réalisée dans différents systèmes d'expression cellulaire, nécessitant la construction de lignées stables de cellules d'insecte et de cellules épithéliales humaines, et leur infection par différents types de vecteurs viraux recombinants, baculovirus ou lentivirus, porteurs du gène rapporteur luciférase. Mais surtout, notre étude a été menée sur des cellules lymphocytaires-T (SupT1), cibles naturelles du virus, exprimant ces αRep et infectées par du VIH-1 naturel infectieux. Nos résultats ont montré que l'αRep4E3 et l'αRep9A8 ont toutes deux un effet négatif significatif sur le cycle réplicatif du VIH-1, mais ciblent des fonctions virales différentes. L'αRep4E3 bloque l'empaquetage du génome viral, tandis que l'αRep9A8 inhibe la maturation et diminue l'infectivité virale. De plus, l'αRep9A8, exprimée de façon constitutive dans les cellules SupT1, leur confère une résistance au VIH: une lignée de SupT1 chroniquement infectée par le VIH-1 a pu être ainsi isolée et maintenue en culture pendant plusieurs semaines, sans effet cytopathique viro-induit apparent. Ces nouvelles données auront des implications non-négligeables dans le choix et la conduite de futures stratégies de thérapie cellulaire anti-VIH. Mots-clés: VIH-1; antivirals; designed molecular scaffolds; HEAT-like repeat; αRep protéine

I 6 3 . 8 . 1 Figure 1 . 1 28 Figure 1 . 2 30 Figure 1 . 3 36 Figure 1 . 4 45 Figure 1 . 5 50 Figure 1 . 6 Figure 1 . 7 Figure 1 . 8 58 Figure 2 . 1 Figure 2 . 2 Figure 2 . 3 Figure 2 . 4 Figure 3 . 1

 638111281230133614451550161718582122232431 Figure 1.1 Schematic overview of the HIV 1 replication cycle 28 Figure 1.2 Diagram of HIV-1 virus particle structure 30 Figure 1.3 Schematic representation of the HIV-1 genome 36 Figure 1.4 Design of the αRep motif 45 Figure 1.5 Structure of filamentous phage 50 Figure 1.6 Structure of baculovirus virions Figure 1.7 Baculovirus life cycle Figure 1.8 Schematic representation of HIV vectors 58

Figure 3 . 2 Figure 3 . 3 Figure 3 . 4 Figure 3 . 5 87 Figure 3 . 6 90 Figure 3 . 7 92 Figure 3 . 8 93 Figure 3 . 9 SDS-PAGE &Western blot analysis of gradient fractions 95 Figure 3 . 10 97 Figure 3 . 99 Figure 3 .Figure 3 .Figure 3 . 14 Figure 3 . 3 Figure 3 .Figure 3 .

 3233343587369037923893399531097399333143333 Figure 3.2 Screening of αRep clones from the third round of panning Figure 3.3 Target-binding activity of αReps against GST-CA21-SP1-NC Figure 3.4 Amino acid sequence, production and purification of αRep4E3 and αRep9A8

Figure 3 .Figure 3 .Figure 3 .Figure 4 . 1

 33341 Figure 3.18 Confocal microscopy of HIV-1 infected, αRep-expressing SupT1 cells Figure 3.19 CA21-SP1-NC full-length bait and deletants Figure 3.20 SDS-PAGE Western immunoblotting of protein pull-down assay representation the binding sites of αRep proteins on the viral target Figure 4.1 Schematic representation of the distinct antiviral effects of αRep4E3 and αRep9A8 in HIV-1 infected SupT1 cell

(Figure 1 . 1 )

 11 . There are many phases in the HIV-1 replication cycle. It begins with the viral entry (attachment and fusion), uncoating, reverse transcription, integration, particle assembly, budding, and maturation. During the virus entry phase, HIV-1 particle attaches to the membrane outside of a CD4+ T cells. Once attached, the virus can inject its genetic material, the viral genomic RNA and viral enzymes into the cells. After that, one of the viral enzymes, the reverse transcriptase (RT) reverse transcribes the viral RNA into the viral DNA with a high proportion of transcription errors. This promotes a high mutation rate during the replication cycle. In the next step, the viral DNA is integrated into the host chromosome by the action of the integrase enzyme (IN), which transforms the cells into a "factory" to produce large quantities of new HIV-1 virions. After virus integrates its genome into the host chromosome, the viral components which are produced by the infected cells move to the inner leaflet of the cell membrane, where they assemble into new virus particles called the viral progeny. The newly formed virus particles then egress from the cell membrane and the maturation phase occurs when the protease (PR) enzyme of the virus cleaves the immature viral structural proteins inside the particle to transform the particles into mature infectious virions. Since there are many crucial steps in the virus life cycle, antiviral drugs or antiviral strategies were designed to target these steps for example, fusion inhibitors, CCR5 inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase strand transfer inhibitors (INSTIs), and protease inhibitors (PIs).

Figure 1 . 1

 11 Figure 1.1 Schematic overview of the HIV-1 replication cycle. The HIV-1 infection begins when the viruses use envelope (Env) glycoprotein spikes bind to CD4 molecule and the co-receptor CC-chemokine receptor 5 (CCR5) (step 1), leading to fusion of the viral and cellular membranes and enter into the cell (step 2). The CA shell uncoating (step 3) facilitates reverse transcription (step 4), which in turn yields the pre-integration complex (PIC). Following import into the cell nucleus (step 5), PIC-associated integrase orchestrates the formation of the integrated provirus, aided by the lens epithelium-derived growth factor (LEDGF), the host chromatin-binding protein (step 6). Proviral transcription (step 7), mediated by host RNA polymerase II (RNA Pol II ) and positive transcription elongation factor b (P-TEFb), generated the different sizes of viral mRNAs, the larger of which require energy-dependent export to leave the nucleus via host protein CRM1 (step 8). The viral protein production (step 9), and genome-length RNA is encapsidated into viral particles with other viral proteins (step 10). Viral-particle budding (step 11) and release (step 12) from the cell is mediated by ESCRT (endosomal sorting complex required for transport) complexes and ALIX and followed by protease-mediated maturation (step 13) to become an infectious viral particle. Each step in the HIV-1 life cycle is a potential target for antiviral intervention165; the sites of action of clinical inhibitors (white boxes) and cellular restriction factors (blue boxes) are indicated. INSTI,

  the infected cells. When more than 2000 molecules of Pr55Gag and 200 Gag-Pol proteins and viral genomes are incorporated. The viral particles still in their immature form then egress from the infected cell. This form of the virus is non-infectious, and is not able to infect the new target cell as the Pr55Gag protein precursors have to be cleaved by the viral PR to generate a set of mature protein. These newly processed proteins then resemble to form mature particle morphology. MA is associated with the inner viral membrane envelope, CA assemble into conical capsid or viral cone, NC is interacted with the viral genomic RNA and packed inside the capsid core (Figure 1.2) together with viral enzymes, reverse transcriptase (RT), integrase (IN).

Figure 1 . 2

 12 Figure 1.2 Diagram of HIV-1 virus particle structure. Top panel: Immature HIV-1 virus particle (left) and mature virus particle (right). Bottom panel: The compositions of HIV-1 Gag polyprotein, MA, CA (CANTD and CACTD), SP1, NC, SP2, and p6 respectively (25). This figure has been obtained from the site: http://www.cell.com/structure/pdf/S0969-2126(10)00355-2.pdf

( 52 )

 52 packed inside. Each RNA strand is approximately 9.7 kilobases (kb) in length. The 5' and 3' ends of the RNA genome are flanked by a long terminal repeat (LTR) promoter region (Figure 1.3). Through specific interactions via packaging signal Ψ, the cis-acting elements, the unspliced genomic RNA is packaged into virions (53). The sequences in genomic RNA that is involved in packaging include stem-loop 3 (SL3) (54) and stem-loop 1 (SL1) (55) in the 5' UTR. Viral RNA once entered into the cytoplasm of the cell, the RT converts the genomic RNA into the viral DNA which is a form ready to integrate into the host chromosome via the action of IN called proviral DNA.

Figure 1 . 3

 13 Figure 1.3 Schematic representation of the HIV-1 genome. The genome of HIV-1 HXB2 (K03455) was used as an example for the HIV-1 genome organization. Open reading frames are shown as rectangles. The start codon of each gene is indicated by the small number in the upper left corner of each rectangle, while the number in the lower right records the last position of the stop codon. For the sequence encoded for pol, the start position is the first T in the sequence TTTTTTAG, which forms part of the stem loop that potentiates ribosomal slippage on the RNA and a resulting -1 frameshift and the translation of the Gag-Pol polyprotein. The tat and rev spliced exons are shown as shaded rectangles. This figure has been obtained from the site: https://www.hiv.lanl.gov/content/sequence/HIV/MAP/landmark.html

  mechanism of action, NRTIs are chain terminators such that once incorporated it will prevent other nucleosides from also being incorporated into the DNA chain because of the absence of a 3' OH group. It acts as a competitive substrate inhibitor. The examples of currently used NRTIs include zidovudine, abacavir, lamivudine, emtricitabine, and tenofovir (64).

12 .

 12 ) Non-nucleoside reverse transcriptase inhibitors Non-Nucleoside reverse transcriptase inhibitor (NNRTI) is a non-competitive inhibitors of reverse transcriptase which inhibit reverse transcriptase by binding to an allosteric site of the enzyme. It can be classified into 2 classes, first generation and second generation NNRTIs. The first generation of NNRTIs are include nevirapine and efavirenz. Meanwhile, the second generation NNRTIs consists of etravirine and rilpivirine (64).

  a high-throughput screen for inhibitors of HIV replication. PF-3450074 and related compounds bind between helix 4 and helix 7 of CANTD. Molecules like the CAP-1, also termed PA-457, and most notably the peptide-based CAI are suitable leader compounds for anti-HIV drug development[START_REF] Braun | HIV-1 capsid assembly inhibitor (CAI) peptide: structural preferences and delivery into human embryonic lung cells and lymphocytes[END_REF]. As the proteolytic processing of Gag is conditioned by the functionality of the viral PR and by the accessibility of its recognition site in Gag, strategies aimed at interfering either with PR enzymatic activity or to reduce access to protein substrate have been considered in the development of anti-HIV-1 drugs. The later concept is represented by a new class of HIV-1 antivirals referred to as maturation inhibitors that have been developed and tested in phase IIb clinical trials. This class of compound is able to inhibit proteolytic maturation of Gag independently of an inhibition of protease activity. The lead drug of this pharmacologic class is betulinic acid (BA), a triterpene compound isolated from the clove-like plant Zygizum cloviflorum. This molecule has been identified as anti-cancer agent. In the last years it has been identified as an anti-HIV-1 molecule. Modification of side chains at position C3 and C28 in the BA scaffold has been used to derive a library of compounds that has been evaluated in vitro for its inhibitory properties. Among these molecules, bevirimat (BVM), potently inhibits HIV-1 replication in tissue culture and is efficacious in HIV-1 infected patients. Biochemical analyses of virus particles grown in the presence of BVM displayed the amount of processed p24 protein decreases in favor of the immature p25 protein reflecting unprocessed CA-SP1 junction. This phenotype is reminiscent of that observed for HIV-1 bearing mutation in CA or at SP1 junction that abolish CA-SP1 processing[START_REF] Sundquist | HIV-1 assembly, budding, and maturation[END_REF][START_REF] Nguyen | The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles[END_REF][START_REF] Dafonseca | The 3-O-(3',3'-dimethylsuccinyl) derivative of betulinic acid (DSB) inhibits the assembly of virus-like particles in HIV-1 Gag precursor-expressing cells[END_REF][START_REF] Dafonseca | The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'dimethylsuccinyl) betulinic acid (DSB) is counteracted by Vif and requires its Zinc-binding domain[END_REF][START_REF] Gonzalez | Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay[END_REF][START_REF] Coric | Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site[END_REF].The combinations of antiretroviral drugs result in the multiple obstacles to HIV-1 to replicate, keep the number of offspring low and reduce the possibility of a superior mutation. In the condition of mutation that conveys resistance to one of the drugs being taken arises, the other drugs will continue to suppress reproduction of that mutation. Combination of antiretroviral drugs usually consist of three drugs from at least two different classes. This three drug combination is commonly known as a triple cocktail. However, major current issues are systemic drug toxicity and the generation of drug resistant viral mutants during prolonged therapy. In addition, viral latency remains an unsolved problem. Patients on antiretroviral therapy remain at increased risk of developing cardiovascular diseases compared to age-matched infected controls, and HIV-1 infection also significantly increases the risk of developing kidney disease, osteoporosis, and number of non-AIDS defining malignancies, even when patients are well controlled on ART[START_REF] Didigu | Gene therapy targeting HIV entry[END_REF][START_REF] Deeks | HIV infection, inflammation, immunosenescence, and aging[END_REF][START_REF] Scherer | Progress and prospects: RNA-based therapies for treatment of HIV infection[END_REF].

  in a right-handed super-helix, giving the image of a solenoid-like structure. The juxtaposition of these α-helices resulting in the extension of the surfaces recruited by evolution as binding sites of their target. In some cases, the local flexibility and the curvature of the elongated solenoid let HEAT repeat to wrap around their targets. Each HEAT repeat protein module (one module = one α-helix pair) contains 37-47 amino acids in length, and present more variable motifs compared to other repeat protein families.In consideration of the known structure of a thermostable HEAT repeat subfamily, their well identified consensus sequence of stable HEAT repeats, the N-cap and the C-caps, this class of HEAT-like protein derived from the phycobylin synthase accessory protein. The library was then constructed as focus on the specific class of HEAT repeat proteins classified as PBS HEATlike repeat by Urvoas A. et al., under the name "αRep" (13), using the rolling circle amplification (RCA) for the generation of gene segments which code for the individual repeat modules. The RCA method is a convenient way to produce large amounts of highly polymerizable repeat sequences and usually used for genome amplification. It has been suggested that RCA can preserve the diversity of complex sequence collections and known as an efficient method to amplify the diversity in the selection experiment such as phage display[START_REF] Burg | Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery[END_REF]. In order to explore the protein sequence space around the conserve residues, protein alignment is the technique that they use to solve the problem. The sequence features of the aligned repeats ensemble is demonstrated in the Figure1.4 (a). In the sequence, some position displays a very high conserve meanwhile some positions are hypervariable. The structure analysis demonstrated the molecular structure of the thermostable HEAT repeat protein. An example αRep protein is made of three consecutive repeats classified as HEAT-PBS repeats. The N-terminal region of the chain is not folded (Figure 1.4 (b)). Structural superposition of an AAA-HEAT repeat and αRep HEAT repeat is showed in Figure 1.4 (c).The structure analysis also reviews that when mapped the αRep sequence onto the guild sequence, Mth187 repeats, the conserve positon can be classified in several group. A set of apolar side chains, the highly conserve R21, the conserve G1, and the most variable positions (Figure1.4 (d-f)).The αRep is a new type of repeat scaffold motif protein in which a library of αRep contains a repertoire of artificial repeat proteins from which specific binding molecules can be selected. Isolation of αRep protein against GFP/EGFP is one model example of using αRep protein as a binder.The study also indicated that the αRep-based GFP binders can be easily produced in the high level and found to be very stable protein. The isolated GFP-specific αReps display high affinity and high specificity which are sufficient for using them as a new tool for purifying GFP-fusion proteins from the whole cell lysate in the protein pull-down assay. Interestingly, expressing αRep inside the cells shows the accurate co-localization with their target protein without causing toxic effects to the cells or induce any protein aggregation. These suggested that αRep protein is a very interesting scaffold protein candidates which can apply to use in many different research fields[START_REF] Chevrel | Specific GFP-binding artificial proteins (alphaRep): a new tool for in vitro to live cell applications[END_REF].

Figure 1 . 4

 14 Figure 1.4 Design of the αRep motif. (a) A Collection of amino acid protein sequences made of consecutive HEAT repeats. The alignments of each repeats are shown in the sequence logo format using the sequence numbering in each motif family as the abscissa. The conserve sequence within the HEAT-PBS repeat subgroup (αRep) and comparison with the previously defined AAA-HEAT repeat subclass (AAA-HEAT). (b) The molecular structure of the thermostable HEAT repeat protein used as a guide (Mth187, PDB code 1TE4). This protein is made of three consecutive HEAT repeats classified as HEAT-PBS repeats. The N-terminal part of the chain is not folded. One HEAT repeat corresponds to two consecutives α-helices (dark gray). (c) Structural superposition of an AAA-HEAT repeat and an αRep HEAT repeat. Residues 396-434 from the PR65/A subunit of human protein phosphatase 2A (dark gray, PDB code 1B3U) 26 and residues

  viruses remain the most frequently targeted, but other viruses from other families were also described. Diverse strategies and targets have been used, providing an incidental demonstration of the versatility of the method. Two main approaches are used: (i) targeting of the viral proteins responsible for viral entry into cells (extracellular steps); (ii) targeting of the various elements of the viral replication complex and the cellular partners of viral proteins within infected cells (intracellular steps)[START_REF] Gonzalez | Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay[END_REF][START_REF] Rakonjac | Filamentous bacteriophage: biology, phage display and nanotechnology applications[END_REF].

Figure 1 . 5

 15 Figure 1.5 Structure of filamentous phage. Composition of phage and arrangement of major coat protein surface domains (92).

  abbreviated AcMNPV. It was the isolate used to develop the original recombinant baculovirus vectors[START_REF] Pennock | Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector[END_REF][START_REF] Smith | Production of human beta interferon in insect cells infected with a baculovirus expression vector[END_REF]) and its genome is still used today as a platform and backbone for the production of most baculovirus vectors today[START_REF] Jarvis | Baculovirus-insect cell expression systems[END_REF]. The AcMNPV contains a 134 kb genome with 154 open reading frames (ORF). The baculovirus particle is formed of a nucleocapsid of 21 nm x 260 nm in size. The baculoviral

Figure 1 . 6 Figure 1 . 7

 1617 Figure 1.6 Structure of baculovirus virions. Schematic representation of the two forms of baculoviruses: (left) the budded virus (BV) and (right) the occluded virus (OCV). BVis the form that released from the cell during the secondary infection. The OCV is the form which is found in the environment, and causes the primary infection in the host. The size of the baculovirus particle is about 21 nm x 260 nm. One single particle consists of the nucleocapsid, for the viral genome coating, the gp64 forming the peplomers, and the membrane lipid bilayer issued from the infected cell. This figure has been obtained from the site: https://en.wikipedia.org/wiki/Baculoviridae

  If a wildtype HIV-1 infect the transduced cells, it would act as a helper virus and rescue the integrated transgene into new viral particles which would spread the transduction beyond the original target cells. The LTRs usually contain three regions: U3, R, and U5. U3 is a viral enhancer/promoter and R in the 3'-LTR is the polyadenylation signal. The mRNA translated from the proviral DNA, does not contain the 5' U3 and 3' U5 elements, but it has the R region caps at both ends of the RNA strand. The duplication of LTR elements occurs during reverse transcription and prior to the integration phase, when U3 in the 3'-LTR is copied and transferred to the 5'-LTR. The first report of this mechanism has been shown for the MLV vector: when part of U3 in the 3'-LTR was deleted, the same deletion was transferred to the 5'-LTR's promoter/enhancer region. This deletion therefore results in transcriptional inactivation of potentially package-able viral genomes in the transduced cell (102). Thanks to this knowledge, the self-inactivating (SIN) mechanism was generated and introduced into HIV vectors (SIN vectors) by deleting the 3'-LTR elements. This SIN modification can reduce the propagation of spontaneously produced replication-competent recombinant HIV-like viruses.The HIV vector has been continuing to improve, and third-generation vectors were later constructed. To increase safety, the Tat protein has been removed and Rev provided from a separate plasmid as shown in Figure1.7 (C). The Tat-independent plasmid is obtained by replacing the U3 promoter region of the 5'-LTR in the transfer vector with the heterologous strong viral promoters from CMV or Rous sarcoma virus (RSV) (103, 104). Four plasmids are used in this version of lentivirus vectors: (i) a packaging construct which carried only gag and pol genes; (ii) a plasmid coding for Rev; an Env (VSV-G) plasmid; and a transfer plasmid with the heterologous promoter. In summary, this third-generation lentiviral vector contains only three genes out of nine genes of HIV. Since the viral genetic material is split into four separate plasmids, there is less possibility for the generating of replicationcompetent HIV-1-like virus.

Figure 1 . 8 1. 2 Objectives 1 . 2 . 1 2 MATERIALS AND METHODS 2 . 1

 182121221 Figure 1.8 Schematic representation of HIV vectors (A) The first generation of HIV vectors includes all of the viral proteins, except the envelope glycoprotein, in a packaging plasmid. VSV-G is provided by a different plasmid. HIV vector plasmid contains LTRs and the transgene is expressed under a strong viral promoter such as the CMV promoter. (B) The second generation of HIV vectors, all of the accessory proteins are removed from the packaging plasmid. Similar to the first generation of HIV vectors, expression of glycoprotein and transgene are provided by different plasmids. (C) The third generation of HIV vectors requires four different plasmids. In addition, to the three plasmids (i.e. the packaging plasmid, Env-encoding plasmid and vector plasmid), Rev protein is provided by a different plasmid. The vector plasmid is also modified by deleting the U3 region from 5'-LTR and partially deleting 3'-LTR to reduce the possible production of replication-competent viruses, and a strong viral promoter such as CMV or RSV is inserted for expression of the vector (52).

2 . 7 of αRep proteins 2 . 7 . 1

 27271 were picked and cultured in 2X YT broth containing ampicillin 100 μg/ml and 1% (w/v) glucose in 96 well plate, 37°C overnight. Then 10 μl of overnight culture were transferred to new plate containing 150 μl of 2X YT broth containing ampicillin 100 μg/ml, tetracycline 10 μg/ml, and 1% (w/v) glucose and cultured at 37°C with shaking for 4 hours. Bacterial cells were infected with 20 MOI of helper phage and incubated at 37°C without shaking for 30 minutes at 37°C and with shaking for 30 minutes. Two hundred microliters of infected cells was transferred to 96-well storage plate containing 1.5 ml of 2X YT broth containing ampicillin 100 μg/ml and kanamycin 70 μg/ml. The plate was covered with gas permeable adhesive seals and cultured at 30°C with shaking for 16-18 hours. Phages in the culture supernatant were collected after the centrifugation and used for phage ELISA to determine their binding activity. Purified GST-CA21-SP1-NC were diluted in coating buffer, then added into a microtiter plate and left for overnight at 4°C in the moisture chamber. The coated wells were washed four times with 0.05% Tween 20 in Tris buffered saline (TBST). The wells were then blocked the non-specific binding with blocking solution (2% BSA in TBST; 200 μl/well) at room temperature for 1 hour with shaking. After the washing step, 100 μl of the culture supernatant of each clone containing phage particles were added and incubated at room temperature for 1 hour. Hundred microliters of mouse anti-M13 conjugated HRP (GE Healthcare Life Sciences) at dilution 1:5,000 in blocking solution were added after the washing step. The wells were then washed again prior adding 100 μl SureBlue™ TMB MicrowellSubstrate (KPL, Gaithersburg, MD) and the optical densities at 450 nm (OD450nm) were measured after adding 1 N HCl. Production Construction of pQE-31 expression vectors for soluble αRep proteins production pQE-31 acceptor vector was used for the production of 6xHis-tagged αRep proteins in M15[pREP4] bacterial cells. The DNA fragments encoding for the αRep proteins were amplified from pHDiExDsbA phagemid vector using a pair of primers; pQE-31_Fw_αRepBamHI (5'-GAG GAG AGG AGA AAT TAA CTA TGA GAG-3') and pQE-31_Rv_αRepHindIII (5'-GAG GAG CTA TCC AGA TGG AGT TCT GAG G-'3') followed by digestions with BamHI and HindIII. The insert fragments were then ligated into linearized pQE-31 vector pretreated with the same cut sites. Subsequently, the recombinant plasmids as shown in the Figure 2.1 were transformed into the XL-1 Blue competent cells. The isolated colonies were picked for colony PCR. The clones which give the PCR product band at correct size were then selected for restriction enzyme analysis using BamHI and HindIII and confirmed by DNA sequencing. The correct plasmid clones were further transformed into M15[pREP4] competent cells for protein expression.

2 . 9 . 1 2 . 9 . 2

 291292 Sf9 stable cell lines stably expressing αRep proteins fused to GFP Construction of pIB vectors expressing (Myr+) αRep4E3-GFP and pIB_(Myr+) αRep9A8-GFP To construct the pIB vector for the expression of the two αRep proteins, (Myr+)αRep4E3-GFP and (Myr+)αRep9A8-GFP, the pIB backbone vector was treated with BamHI and HindIII restriction enzymes. The insert fragments of these two genes were obtained from pQE-31-αRep4E3 and pQE-31-αRep9A8 by PCR technique using a pair of specific primers, pIB_Fw_(Myr+) αRep (5'-GAG AAG CTT ATG GGA TCC AGA AAA-3') and pIB_Rv_αRep (5'-GAG GAG AAG CTT TTA TGG GCT-3') then treated the PCR products with the same cut sites as backbone vector. The ligation was performed using T4 DNA ligase and the ligation products were transformed into XL-1 Blue competent cells followed by plating the cells on the LB agar containing 100 μg/ml of ampicillin. Isolated colonies were picked for colony PCR and restriction enzymes analysis. The positive clones were confirmed by DNA sequencing. Establishment of Sf9 cells stable expressing (Myr+) αRep4E3-GFP and pIB_(Myr+) αRep9A8-GFP Aliquots of Sf9 cells were transfected with 2.5 μg pIB-based vectors (Thermo Fisher Scientific, Waltham, MA) encoding the myristoylated αRep protein fused to green fluorescence protein, pIB_(Myr+) αRep4E3-GFP and pIB_(Myr+) αRep9A8-GFP, using the DOTAP Liposomal Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany) as protocol directed. The transfected cells were maintained in complete Grace's insect medium containing 100 μg/ml blasticidin (Thermo Fisher Scientific, Waltham, MA) to isolate the blasticidin resistant cell lines. Two cell lines were generated, Sf/(Myr+)αRep4E3-GFP and Sf/(Myr+)αRep9A8-GFP. The (Myr+) αRep4E3-GFP and (Myr+) αRep9A8-GFP protein expression was monitored under fluorescence microscope and western blotting analysis using rabbit polyclonal anti-αRep protein followed by phosphatase-labeled anti-rabbit monoclonal antibody. 2.10 Luciferase-based quantitative assay of αReps on HIV-1 VLP assembly Sf9-derived cell lines, Sf/(Myr+)αRep4E3-GFP and Sf/(Myr+)αRep9A8-GFP were seeded on 6-well plate to have 70-80% confluent then infected with the recombinant baculovirus carrying gene encoded HIV-1 Gag polyprotein (AcMNPV gag ), or coinfected with AcMNPV gag and HIV-1 auxiliary protein Vpr fused to the luciferase reporter gene (AcMNPV lucvpr ) at multiplicity of infection (MOI) 10 PFU/cell. At 48 hours post-infection the viral-like particles (VLPs) produced from the cells in culture supernatant as shown in Figure 2.2 were collected isolated by 30%-50% sucrose gradient ultracentrifugation. After ultracentrifugation in an SW41 Ti Swinging-Bucket Rotor at 28,000 rpm, the membrane-enveloped VLP pellet were resuspended in lysis KDT buffer (0.1 M potassium phosphate buffer, pH 7.8, 1 mM DTT, containing 0.2% Triton X-100) for 30 minutes at 37°C with vortexing every 10 minutes. Enzymatic assay of VLP-associated luciferase activity were then measured after adding luciferase substrate solution (0.1 M Tris acetate pH 7.8, 1 M DTT, 0.5 M EDTA, 1 M MgSO4, ) to quantify the level of VLP production using a Lumat LB-9501 luminometer (Berthold Technologies, Bad Wildbad, Germany).

Figure 2 . 2

 22 Figure 2.2 Luciferase based VLP assembly assay. After double infection αRep expressing cells with two recombinant baculoviruses, AcMNPV gag and AcMNPV lucvpr , VLPs carrying the Luc-Vpr fusion protein are released in the culture supernatant and can be isolated by conventional centrifugation methods or by isopicnic ultracentrifugation in 30%-50% sucrose-D2O density gradient at 48 hours post-infection and assayed for luciferase activity.

Figure 2 . 3

 23 Figure 2.3 The schematic figure of CGW lentiviral transfer vectors construction for stable expression of αRep proteins in target cells. The DNA sequences coded for αRep proteins were inserted into CGW lentiviral vector using EcoRI and BstXI cloning sites.

  minutes. Viral vector pellets were resuspended in sterile PBS (EMD Millipore Corp., Billerica, MA) containing 0.1% Bovine Serum Albumin (BSA) (EMD Millipore Corp., Billerica, MA), split into 50 μl aliquots, and kept at -80˚C. The viral titer were determined by the percentage of EGFP positive cells by infection of HEK293T cells with serial dilution of the samples at 48 hours post-transduction. The equation for calculation the titer is described below.

  SupT1 cell lines stably expressing αRep proteinsAliquots of SupT1 cells were transduced with 1 MOI of the VSV-G-pseudotypedCGW_αRep-EGFP vectors by spinoculation at 2,500 x g, 32 °C for 2 hours in growth medium containing 8 μg/ml of Polybrene (Sigma-Aldrich, St. Louis, MO). At 16 hours post-transduction cells were washed three times using serum free medium then maintained at 37°C and 5% CO2 in fresh C-RPMI supplemented with 10% FBS, penicillin 100 U/ml, and streptomycin 100 μg/ml. The cells were further maintained in fresh C-RPMI medium and divided every 3 days. The transduction efficiency and stability were monitored by fluorescence microscopy and flow cytometry.

Figure 2 . 4

 24 Figure 2.4 Production of VSV-G pseudotyped lentiviral vector particles. VSV-G pseudotyped lentiviral vector particles carrying αRep gene was produced from HEK293T cells through transient co-transfection of vectors which encode genes for the viral components of including (1.) packaging vector (pMDLgag/polRRE), (2.) Rev expressing vector (pRSV-Rev), (3.) Envelope vector (pMD.2G) VSV-G, and (4.) CGW transfer vector. Pseudotype lentiviral vector particles were then harvested from the culture medium and concentrated by 20% sucrose cushion ultracentrifugation. The viral vector titer was determined by transduction into HEK293T cells following analysis by flow cytometry.

  6 and incubated overnight at 4°C in a moisture chamber. The anti-MA antibody, hybridoma clone MHSVM33C9/ATCC HB-8975, was obtained from the American Type Culture Collection (ATCC, Manassas, VA). It is known to recognized the epitope (DTGHSSQVSQNY) located at the C terminal MA of Gag polyprotein (107, 108). The coated wells were washed 3 times with PBST and blocked with 200 μl of blocking solution (2% BSA PBS) at room temperature for 1 hour. After the washing step, 60 μl culture supernatant from each infected SupT1 expressing Rep-EGFP was normalized the p24CA titers to final concentration 80 μg/ml. The samples were then pre-incubated with 1% Triton X-100 at room temperature for 10 minutes and 10 ng/ml of biotinylated synthetic p17MA C-terminal peptide were added to the well and incubated for 1 hour.

Figure 2 . 5

 25 Figure 2.5 CA21-SP1-NC full-length bait and deletants. From top to bottom: full-length target CA21-SP1-NC; deletion of the C-terminal Zinc finger (ΔZF2); deletion of the two Zinc fingers ZF1 and ZF2.

  -NC is our privileged target for screening the αRep library to obtain a new family of HIV-1 assembly inhibitors. The protein interactors of SP1-NC could have several effects: (i) on the Pr55Gag structure and oligomerization process; (ii) on the viral protease activity and the blockage of the maturation cleavage of CAp25 into CAp24-SP1, viral genome packaging and the infectivity of the virus. We have chosen the polyprotein domain of Pr55Gag between L343 and F433 (91 residues) overlapping the C-terminal 21 residues of the CA protein, the SP1 linker and the region of the NC protein encompassing its two zinc fingers (abbreviated CA21-SP1-NC) as the bait for our selection of αRep molecules. The NMR structure of SP1-NC-SP2 was shown in the Figure 3.1 to elucidate the folded structure of this protein domain.

Figure 3 . 1

 31 Figure 3.1 Amino acid sequences and the NMR structure of the SP1-NC-SP2 domain of HIV-1 Gag polyprotein. Top panel shows the sequence of amino acids of NC containing the two zinc finger motive CCHC and SP2. Bottom panel is the 3D structure of NC-SP1 as determined by NMR. The position of individual amino acid are indicated in the number, zinc ions are showed in the yellow round symbol.

Figure 3 . 2

 32 Figure 3.2 Screening of 30 αRep clones from the third round of panning. Phages obtained from single individual clones were used to determine the high affinity binders against the GST-CA21-SP1-NC target protein. GST served as a negative control for non-specific

  sequencing. The αRep proteins were then expressed in E. coliM15 [pREP4]) and purified by affinity chromatography using HisTrap columns. The soluble form of the six selected αReps were analyzed for their binding activity to the viral target in vitro, using indirect ELISA technique. Microtiter ELISA plates were coated with GST-CA21-SP1-NC, the αRep proteins were added individually to the well, and their binding activity was detected by using a rabbit polyclonal anti-αRep antibody followed by peroxidase-conjugated goat anti-rabbit antibody. The results were shown in Figure3.3 A. Anti-GST antibody was used to confirm that target GST-CA21-SP1-NC and control GST were successfully immobilized onto the micro-well plates (Figure3.3 B). Distinct binding activities between the αReps clones were revealed when the wells were coated at 5 μg/ml GST-CA21-SP1-NC, compared to wells coated with GST proteins. The αRep 4E3 clone showed the highest binding activity, and 9A8 the second highest. The other four clones showed a lower binding activity to the target protein (Figure3.3 A). This has already been observed for certain phage-displayed proteins, whose binding properties change with their environment. Within the gp3 fusion structure, their binding interfaces can be significantly altered, positively or negatively. This seemed be the case for αRep clones 4D4, 4F2, 5A12, and 5F1 which behaved differently in fusion with phage gp3 protein (strong binders), and in soluble form (moderate binders). Thus, only the two αReps, αRep4E3 and αRep9A8, were selected for further characterization in the next experiments.

Figure 3 . 3

 33 Figure 3.3 Target-binding activity of αReps against GST-CA21-SP1-NC. Binding activity of soluble αRep clones were measured by ELISA. A, Binding of αReps to the viral target GST-CA21-SP1-NC (black bars), with GST used as a background control (grey bars). Target protein and control GST protein at 5 μg/ml were immobilized on the plate. Soluble purified αRep clones were used at 10 μg/ml, and the level of αRep binding was assayed using specific anti-αRep antibody. B, Anti-GST antibody was used to perform the quality control of the coated plates in terms of target protein immobilization.

Figure 3 . 4

 34 Figure 3.4 Amino acid sequence, production and purification of αRep4E3 and αRep9A8. A & C, Amino acid sequences of αRep4E3 and αRep9A8, with each of amino acid indicated in one single-letter code. Constant regions of the proteins are in black letters, whereas variable residues are shown in red. An oligo-His tag is fused to the N-terminus of each protein. The multiple repeat motifs are stacked between N-cap and C-cap. B & D, SDS-PAGE analysis of αRep4E3 and αRep9A8 purified by affinity chromatography on Ni-NTA agarose resin. WCL, whole cell lysate before αRep purification, containing all soluble proteins.

  fluorescent protein (GFP) encoded gene and to the coding sequence of the oligo-Histidine tag at their C-terminus. In parallel, the N-terminus of both proteins was modified to become a substrate for cytoplasmic N-myristoyl-transferase. This design resulting in the co-translational amidification of the alpha-amino group of the N-terminal glycine residue by the myristic acid, and the attachment of a myristoyl group to the N-terminus of αRep4E3 and αRep9A8 via a covalent amide bond (110). The aim of adding the N-myristoylation to the αRep proteins was to address them to the inner leaflet of the cell plasma membrane, where the VLP assembly occurs. The schematic diagram of the construction of these modified αRep-GFP genes is shown in Figure 3.5 (A). The gene constructs were cloned into the pIB/V5-His vector which contained a blasticidin resistance gene. The recombinant plasmids were then performed the DNA sequencing analysis. The correct DNA sequence of the constructs were transferred to Sf9 cells by liposome-mediated transfection, to generate the Sf/(Myr+)4E3-GFP and Sf/(Myr+)9A8-GFP cell lines. Cells were treated with blasticidin, a potent translational inhibitor, to eliminate the parental Sf9 cells and only maintain in culture those cells which have received the recombinant plasmid carrying the blasticidin-resistant gene, and which stably expressed the αRep-GFP molecules. In these two cell lines, the αRep4E3-GFP and αRep9A8-GFP genes were integrated into the cellular genome resulting in cells stably expressing the αRep-GFP proteins under the control of the baculovirus immediateearly promoter, OplE2 promotor. The expression of (Myr+)αRep4E3-GFP and (Myr+)αRep9A8-GFP proteins was successfully produced intracellularly as monitored by fluorescence microscopy as shown in the Figure 3.5 B. The top panel represented the Sf9 cells expressing the (Myr+)αRep4E3-GFP and the bottom panel shows the protein expression of (Myr+)αRep9A8-GFP. Each panel displays the phase contrast and GFP signal on the left and right hand side respectively.

Figure 3 . 5

 35 Figure 3.5 Construction of Sf9 cells stably expressing N-myristoylated αRep4E3-GFP and N-myristoylated αRep9A8-GFP. A, Schematic diagram of the αRep genes designed for stable expression of the N-myristoylated αRep4E3 and αRep9A8 proteins in Sf9-derived cells [Sf/(Myr+)4E3-GFP and Sf(Myr+)9A8-GFP]. The genes coding for the αRep proteins were modified to carry the sequence MG2AAA at their N-terminus, andGFP and His6-tag at their C-terminus respectively. The peptide MG2AAA is a substrate for N-myristoyl-transferases. In this enzymatic process, methionine-1 is removed and the new N-terminus of glycine at position 2 is the receiver of the N-myristoyl group.

Figure 3 . 6

 36 Figure 3.6 Biological effects of αRep4E3 and αRep9A8 on HIV-1 VLP assembly.

3 . 4 . 3

 343 antibody. C-D, Luciferase assays. The peaks of luciferase activity correlate with the levels of VLPs released from C control Sf9 cells, or D from Sf/(Myr+)4E3-GFP or Sf/(Myr+)9A8-GFP cells. Biological effects of αRep4E3 and αRep9A8 on HIV-1 VLP production in heterologous system: qualitative aspects Although the VLP production by AcMNPV gag -infected insect cells was not influenced by the co-expression of αRep4E3 or αRep9A8, it was important to verify their quality in terms of size, morphology and internal structure. Ultrathin sections of AcMNPV gag -infected Sf/(Myr+)4E3-GFP and Sf/(Myr+)9A8-GFP cells were analyzed under the electron microscope (EM) at 48 hours post-infection, in comparison with control AcMNPV gag -infected Sf9 cells, with a special focus on the plasma membrane and VLPs budding into the extracellular milieu. Both αRep4E3 and αRep9A8 altered the morphogenetic pathway of VLPs, resulting in the assembly and extracellular release of aberrant forms, but with significant differences between αRep4E3 and αRep9A8.

Figure 3 . 7

 37 Figure 3.7 Morphological analysis of VLPs from αRep4E3-expressing insect cells. Electron micrographs of ultrathin sections of AcMNPV gag -infected cells, showing VLPs budding from their plasma membrane. A, control Sf9 cells; B-F, Sf/(Myr+)αRep4E3-GFP cells. Cells were infected with recombinant baculovirus AcMNPVgag, harvested at 48 hours pi, and samples processed for EM observation. Note the number of aberrant VLPs in panels B-F, contrasting with the regular shape of VLPs from control Sf9 cells, shown in panel A.

Figure 3 . 8 3 . 4 . 4

 38344 Figure 3.8 Morphological analysis of VLPs from αRep9A8-expressing insect cells. Electron micrographs of ultrathin sections of AcMNPV gag -infected cells, showing VLPs budding from their plasma membrane. A, Control Sf9 cells; B-G, Sf/(Myr+)αRep9A8-GFP cells. Cells were infected with recombinant baculovirus AcMNPVgag, harvested at 48 hours pi, and samples processed for EM observation. A, VLPs from control Sf9 cells showed a regular spherical shape. Aberrant VLPs are presented in panels B-D. Note the abnormal accumulation of Pr55Gag polyprotein at the plasma membrane, visible in panels E-G.

Figure 3 . 9

 39 Figure 3.9 SDS-PAGE &Western blot analysis of gradient fractions. Sf/(Myr+) αRep4E3 and Sf/(Myr+)αRep9A8 cells were infected with AcMNPV gag . At 48 hours pi, culture media were analyzed by ultracentrifugation through a sucrose-D2O density gradient, and gradient fractions probed with anti-Gag and anti-His tag antibodies. The fractions containing VLPs and corresponding to the relative density ρ = 1.18 are indicated by an accolade.

Figure 3 . 3 . 6

 336 Figure 3.10 Confocal microscopy of HeLa cells expressing αReps. The top and bottom rows of panels show HeLa cells transfected with CGW_αRep4E3-GFP and CGW_αRep9A8-GFP vectors, respectively. The columns (starting from left to right) show cell samples in phase contrast (DIC), the EGFP signal, the 4,6-diamidino-2-phenylindole (DAPI) signal, and the merging of the fluorescent DAPI and EGFP images, respectivel

Figure 3 . 1 infection 3 . 7 . 1

 31371 Figure 3.11 Fluorescence microscopy and flow cytometry analysis of SupT1 cells transduced by a lentiviral vector. A, The cell morphology and expression of EGFP expression were monitored by light and fluorescence microscopy, using an inverted fluorescence microscope at 100X magnification. B, Flow cytometry analysis of

  the expression of antiviral αRep molecules throughout the time of the HIV-1 challenging experiment, the percentage of EGFP-positive cells was controlled by flow cytometry at each time pi. As presented in Figure3.11 C, our stable SupT1 cell lines stably expressed αRep proteins at a level close to 100%.

Figure 3 . 1 (

 31 Figure 3.12 αRep4E3 and αRep9A8-mediated protection of SupT1 cells against HIV-1 challenge. A, Expression of EGFP in HIV-1 infected SupT1 cells and SupT1 expressing αRep-EGFP over time. The data presented were the average from triplicate experiments. Bars represent mean ± SD (n=3). B, SupT1 cells stably expressing αRep-EGFP proteins were infected with HIV-1 at a multiplicity of infection of 1 (MOI 1). Culture supernatants were collected at day 7, 14, and 21 pi, and the levels of CAp24 antigen determined by enzyme-linked immunosorbent assay. C, The morphology of the infected cells was monitored the same day as the assays for extracellular CAp24.

3. 7 . 4

 74 Cell viability and αRep protein expression in HIV-1 infection SupT1 cellsHIV-1 infection generally induces a marked cytopathic effect characterized by the occurrence of giant cells or syncytia. The change in cell morphology and viability is a reliable indicator of the level of cellular infection. Taking this point into consideration for our HIV-1 challenge assay, our different types of modified or non-modified SupT1 cells, with or without αRep protein expression, were infected with HIV-1 at the same MOI, and the cell cultures maintained for one month. Every three days, the cells were collected and controlled for morphology, viability and numeration. The results showed that both cell count and viability decreased with time during HIV-1 infection (Figure3.15 A & B), except for the early phase (D3-D7 pi). During this phase, the cell concentration increased (Figure 3.15 A), because the virus does not cause much effect on cellular homeostasis early in infection. Unexpectedly however, there was a rebound of cell growth for the SupT1/αRep9A8-EGFP cells on D28-D38 pi, shown by both cell count and cell viability assays. In addition, these cells looked perfectly healthy under the light and fluorescence microscope. The extracellular medium of SupT1/αRep9A8-EGFP cells at D38 was also investigated for the Cap24 level and viral genomes. The result showed that culture supernatant contained viral particles and viral genomes (Figure 3.15 D & E), at the level similar to those measured at D14 pi. Of note, these concentrations were respectively 10-fold lower for CAp24 (Figure 3.12 B) and lower for viral genome copies (Figure 3.13), compared to those of control cell media at the same time point. More interestingly, the viral infectivity assays showed that the extracellular HIV-1 particles released by SupT1/αRep9A8-EGFP at D38 pi were noninfectious (Figure 3.15 F).

  to genome encapsidation; (ii) whereas no living cells were detectable in cultures of control SupT1 cells or SupT1/αRep4E3-EGFP cells infected with HIV-1 at D21-D28 pi, while αRep9A8-expressing SupT1 cells regained viability at later times pi (D38).

Figure 3 .

 3 Figure 3.15 Total cell number, cell viability and cell morphology after challenge with HIV-1NL4-3. (A) Total cell number per mL; (B) percentage of cell viability at different days pi. Bars represent the mean ± SD (n=3). (C) Light and fluorescence microscopy analysis of SupT1αRep9A8 cells at D38 pi, using an inverted fluorescence microscope at 100× magnification. (D) Comparison of the CAp24 levels in the cell culture medium of HIV-1 infected SupT1/αRep9A8-EGFP cells at D14 and D38 pi. (E) Concentrations of viral genomes in the cell culture medium of HIV-1 infected SupT1/αRep9A8-EGFP cells at D14 and D38 pi. (F) Infectivity titers of viral particles present in the cell culture medium of HIV-1 infected SupT1/αRep9A8-EGFP cells at D14 and D38 pi, expressed as the percentage of GFP-positive Jurkat-GFP reporter cells.

Figure 3 .

 3 Figure 3.16 Maturation cleavage of Pr55Gag in viral particles released by different cell types. Control SupT1 cells, SupT1αRep9C2, SupT1αRep4E3 and SupT1αRep9A8were infected with HIV-1, and extracellular media were collected. After normalization of the CAp24 level (80μg in all samples), the degree of PR-mediated cleavage of Pr55Gag was determined using our ELISA-based maturation assay (Refer to Materials & Methods). The percentage of %inhibition or %relative viral maturation obtained in this assay is directly related to the percentage of maturation of extracellular viral particles, and therefore their degree of infectivity. Results presented are from triplicate experiments (mean ± SD; n=3). Note the lower degree of maturation of viral particles issued from SupT1αRep9A8 cells, compared to SupT1αRep4E3 and to control cells, SupT1 and SupT1αRep9C2, which all three show similar levels of particle maturation.

Figure 3 .

 3 Figure 3.18 Confocal microscopy of HIV-1 infected, αRep-expressing SupT1 cells. A, HIV-1-infected control SupT1 cells and B, αRep-expressing SupT1/αRep9C2-EGFP, C, SupT1/αRep4E3-EGFP, and D, SupT1/αRep9A8-EGFP cells, were collected at D10 pi, fixed, permeabilized, and reacted with Qdot705-conjugated anti-CAp24 monoclonal antibody. Nuclei were counterstained with DAPI. A-D, Individual images obtained with the excitation wavelength 405 nm (DAPI), 488 nm (EGFP), and 561 nm (Qdot705), respectively, are presented as vignettes on the left side of each panel; merged images of EGFP and Qdot705 signals are shown on the right side. Enlargement of C and D showing the colocalization of αRep and Gag proteins (white arrows).

  target was first investigated by ELISA, using glutathione-coated microplates and surfaceimmobilized GST-fused full-length target and carboxy-terminal deletion mutants of CA21-SP1-NC. In this experiment protein pull-down assays were then carried out to identify the biding domain of each individual αRep on their target protein. The glutathione-coated beads were added to the mixture of αRep4E3 or αRep9A8 pre-incubated with the viral target protein, GST-CA21-SP1-NC or its different C-terminal deletants (Figure3.19 A-B). The results showed that both αRep4E3 and αRep9A8 bound equally well to the full-length target CA21-SP1-NC, but that the deletion of the two zinc fingers, as in GST-CA21-SP1, completely abolished their binding. However, deletion of the second zinc finger (ΔZF2), as in GST-CA21-SP1-NCΔZF2, did not modify the binding pattern of αRep9A8, whereas it decreased the αRep4E3 binding (Figure3.20 A). This result suggested that the binding site of αRep9A8 was restricted to the ZF1 domain, since αRep9A8 binding to the viral target was not influenced by ZF2. By contrast, the αRep9A8 binding site apparently overlapped both ZF1 and ZF2, since after deletion of ZF2, attachment of αRep4E3 to the viral target still persisted, implying the occurrence of another binding site on ZF1 (Figure3.20 B). Of note, no essential binding determinant for αRep4E3 and αRep9A8 could be detected on the SP1 linker domain, using these pulldown assays.

Figure 3 .

 3 Figure 3.19 CA21-SP1-NC full-length bait and deletants. A, Amino acid sequences of the domains of HIV-1 Gag precursor (L343-F433; HIV-1 LAI isolate) used as gthe viral bait of the αRep screen. The residue of the SP1 domain are in bold; the cysteine and histidine residues of the zinc finger (ZF1 and ZF2) responsible for the Zn coordinates are in red; the residues of the basic motif separating ZF1 and ZF2 are in blue. B, Schematic representation of GST-fused viral construct. From top to bottom: full-length target CA21-SP1-NC; deletion of the C-terminal Zinc finger (∆ZF2); deletion of the two Zinc fingers ZF1 and ZF2.

Figure 3 .

 3 Figure 3.20 SDS-PAGE Western immunoblotting of protein pull-down assay representation the binding sites of αRep proteins on the viral target. A, Left panel: The glutathione-coated beads were added to the mixture of αRep4E3 or αRep9A8 preincubated with the viral target protein, GST-CA21-SP1-NC or its different C-terminal deletants. The pull-down phases were analyzed by SDS-PAGE and immunoblotting, using polyclonal anti-αRep and anti-GST monoclonal antibody respectively. Right panel: summary of the binding site of the αRep4E3 and αRep9A8 on their viral target. B, The schematic represented the hypothesis of the biding site of αRep4E3 and αRep9A8.

Figure 4 . 1 .Figure 4 . 1

 4141 Figure 4.1 Schematic representation of the distinct antiviral effects of αRep4E3 and αRep9A8 in HIV-1-infected SupT1 cell.The normal pathway of HIV-1 assembly and egress is represented in(1); the molecular mechanisms of the αRep4E3-and αRep9A8mediated antiviral effects are displayed in pathways (2) and (3). αRep4E3 (blue symbol) negatively interferes with Gag-RNA interaction, genome packaging and promotes late phase reverse transcription and viral DNA packaging; αRep9A8 (yellow symbol) acts as a maturation inhibition

  to 10 ml and boiled in boiling water. Filtered through 0.2 μm Millipore filter and stored at 4 in 100 ml distilled water Sterilized by autoclave, and kept at 4 °C in 1,000 ml distilled water.Sterilized by autoclave, poured on Petri dish (plate) and stored at 4 to 10 ml and boiled in boiling water. Filtered through 0.2 μm Millipore filter and stored at 4 and adjusted pH into to 7.05 with NaOH Added volume with ddH20 into 500 ml. Filtered through 0.2 μm Millipore membrane filter.

  การติ ดเชื อไวรั สเอชไอวี 1 ก่ อให้ เกิ ดโรคติ ดเชื อเรื อรั งที ต้ องรั กษาตลอดชี วิ ต นอกเหนื อไปจาก การรั กษาด้ วยยาต้ านไวรั สซึ งเป็ นการรั กษามาตรฐานในปั จจุ บั นแล้ ว การรั กษาด้ วยยี นถื อเป็ นอี กหนึ ง แนวทางใหม่ ที ช่ วยเพิ มความหวั งในการรั กษาให้ ดี ขึ น โดยทํ าการออกแบบยี นที กํ าหนดการสร้ าง โมเลกุ ลที มี ประสิ ทธิ ภาพในการยั บยั งการแบ่ งตั วของเชื อไวรั ส ในการศึ กษานี เป็ นครั งแรกที ใช้ ยี น สํ าหรั บสร้ างโปรตี นแอลฟารี พี ทซึ งเป็ นโปรตี นโครงสร้ างพิ เศษที แตกต่ างไปจากแอนติ บอดี มาทดสอบการยั บยั งวงจรชี วิ ตของไวรั สเอชไอวี 1 โดยทํ าการคั ดเลื อกโปรตี นแอลฟารี พี ทที สามารถ จั บได้ กั บบริ เวณนิ วคลี โอแคพซิ ด ซึ งเป็ นส่ วนหนึ งของโปรตี นโครงสร้ างของเชื อไวรั สโดยเทคนิ ค ฟาจดิ สเพลย์ พบว่ าสามารถคั ดเลื อกโปรตี นแอลฟารี พี ทโคลนที จั บอย่ างจํ าเพาะได้ 2 โคลนคื อ สี อี สาม (αRep4E3) และเก้ าเอแปด (αRep4E3) ซึ งโปรตี นแอลฟารี พี ทดั งกล่ าวนี ประกอบไปด้ วย จํ านวนรี พี ท 7 รี พี ท มี ขนาดโมเลกุ ล 32 กิ โลดาลตั น และ 6 รี พี ท มี ขนาดโมเลกุ ล 28 กิ โลดาลตั น ตามลํ าดั บ โปรตี นทั งสองโมเลกุ ลมี ระดั บการแสดงออกที สู งและมี ความคงตั วสู งในเซลล์ หลายชนิ ด ทั งในแบคที เรี ย เซลล์ แมลง และเซลล์ มนุ ษย์ จากนั นนํ ายี นของโปรตี นทั งสองโคลนนี เข้ าสู ่ เซลล์ เพื อ ทดสอบฤทธิ ในการยั บยั งวงจรชี วิ ตของเชื อไวรั สภายในเซลล์ ทั งในเซลล์ แมลงชนิ ดเอสเอฟเก้ า (Sf9) และในเซลล์ เม็ ดเลื อดขาวของมนุ ษย์ ชนิ ดที เซลล์ (SupT1)ซึ งการทดสอบผลของแอลฟารี พี ททั งสอง ในการรบกวนการผลิ ตอนุ ภาคเสมื อนไวรั ส (VLP) โดยใช้ เซลล์ แมลงนั น ได้ ทํ าการออกแบบให้ โปรตี นแอลฟารี พี ทมี การแสดงออกในรู ปแบบที มี ไมริ สติ กแอซิ ดติ ดอยู ่ ด้ านทางปลายอะมิ โนและ เชื อมต่ อโปรตี นเรื องแสงสี เขี ยว (GFP) เข้ าไปทางด้ านปลายคาร์ บอกซิ ลิ ค จากนั นจึ งติ ดเชื อเซลล์ ดั งกล่ าวด้ วยแบคคู โลวั สไวรั ส AcMNPV gag ที ทํ าหน้ าที ในการนํ าส่ งยี นกํ าหนดการสร้ างโปรตี นแกก (Gag) ของไวรั สเอชไอวี 1 และติ ดตามผลการรบกวนการสร้ าง VLP ในเซลล์ ดั งกล่ าว จากผลการ ทดลองพบว่ าโปรตี นแอลฟารี พี ททั งสองมี ผลทํ าให้ การกระกอบอนุ ภาคของไวรั สแปรเปลี ยนไปใน เชิ งคุ ณภาพ พบรู ปแบบที แตกต่ างกั นทางสั ณฐานวิ ทยาของ VLP ซึ งเป็ นผลมาจากฤทธิ ของโปรตี น แอลฟารี พี ทเมื อศึ กษาโดยใช้ กล้ องจุ ลทรรศน์ อิ เล็ กตรอน โดยสั งเกตพบว่ า αRep4E3 มี ฤทธิ ส่ งเสริ ม ให้ มี การผลิ ต VLP ที มี รู ปร่ างผิ ดปกติ ในขณะที αRep9A8 มี ผลทํ าให้ เกิ ดการสะสมของโปรตี น Gag ที ผนั งเซลล์ Sf9 และพบอี กว่ าโปรตี นแอลฟารี พี ททั งสองถู กบรรจุ เข้ าไปในอนุ ภาค VLP จากนั นจึ ง ทํ าการศึ กษาฤทธิ ของโปรตี นแอลฟารี พี ทต่ อในเซลล์ เม็ ดเลื อดขาวของมนุ ษย์ ชนิ ด SupT1 โดยได้ ออกแบบโปรตี นแอลฟารี พี ททั งสองให้ มี การแสดงออกภายในเซลล์ ได้ อย่ างต่ อเนื องโดยได้ เชื อม EGFP ไว้ ทางด้ านปลายปลายคาร์ บอกซิ ลิ คของโปรตี น จากนั นทํ าการติ ดเชื อเซลล์ ที มี การแสดงออก ของโปรตี นแอลฟารี พี ททั งสองชนิ ดด้ วยไวรั สเอชไอวี ที 1 สายพั นธุ ์ NL4-3 ที 1 MOI ติ ดตามผลการ ทดลองพบว่ าโปรตี นแอลฟารี ทั งสองตั วมี ความสามารถในการต้ านการเพิ มจํ านวนของไวรั สและพบ อี กว่ าโปรตี นแอลฟารี พี ททั งสองออกฤทธิ ในช่ วงท้ ายของวงจรชี วิ ตของเอชไอวี โดยที ไม่ มี ผลในการ ยั บยั งการฝั งตั วของดี เอ็ นของไวรั สในโครโมโซมของเซลล์ ติ ดเชื อ นอกจากนั นพบว่ าไวรั สตั วใหม่ สร้ างได้ น้ อยลงและช้ าลงในเซลล์ ที มี การแสดงออกของโปรตี นแอลฟารี พี ททั งสอง และยั งพบอี กว่ า กลไกในการยั บยั งไวรั สนั นแตกต่ างกั นระหว่ างโปรตี นแอลฟารี พี ททั งสองตั ว โดย αRep4E3 มี ฤทธิ ในการรบกวนการบรรจุ อาร์ เอ็ นเอจี โนมเข้ าไปในอนุ ภาคไวรั ส ในขณะที αRep9A8 พบว่ ามี การ รบกวนกระบวนการสร้ างไวรั สที สมบู รณ์ (maturation)โดยทํ าหน้ าที เสมื อนเป็ นตั วขั ดขวางเอ็ นไซม์ โปรติ เอสไม่ ให้ สามารถเข้ าตั ดโปรตี น Gag ได้ ซึ งขั นตอนดั งกล่ าวนี มี ความจํ าเป็ นสํ าหรั บการผลิ ต ไวรั สตั วเต็ มวั ยตั วใหม่ อี กทั งยั งพบประเด็ นที น่ าสนใจว่ าเซลล์ SupT1 ที มี การแสดงออกของ αRep9A8 สามารถรอดชี วิ ตนานถึ ง 38 วั นในสภาวะที เป็ นการติ ดเชื อเรื อรั ง พบการสร้ างไวรั สตั วไม่ เต็ มวั ยและไม่ มี ความสามารถในการติ ดเชื อออกมาในนํ าเพาะเลี ยงในระดั บตํ า จากการศึ กษาทั งหมดนี สามารถกล่ าวได้ ว่ าโปรตี นแอลฟารี พี ทเป็ นโปรตี นชนิ ดใหม่ ซึ งถื อเป็ นทางเลื อกที น่ าสนใจสํ าหรั บการ นํ ามาใช้ ในการพั ฒนาเป็ นโมเลกุ ลต้ านไวรั สและสามารถนํ ามาต่ อยอดเพื อการพั ฒนาเซลล์ ต้ านไวรั ส เอชไอวี เพื อใช้ ในการรั กษาได้

Keywords: HIV-1; antivirals; designed molecular scaffolds; HEAT-like repeat; αRep proteins บทคั ดย่ อ

1 Literature review 1.1.1. Human immunodeficiency virus (HIV)

	SP1 domain has been shown to be involved in the correct particle assembly. Mutation in
	the key amino acid residues of SP1 especially the first four amino acids can alter the viral
	morphology and interfere the virus infectiveness. The NC region is essential for the viral
	genomic RNA packaging and promote Gag-Gag interaction. As mentioned, our target
	protein CA21-SP1-NC is critical in the process of Pr55Gag assembly and genomic RNA
	packaging in HIV-1-infected cells, we therefore assumed that expressing αRep proteins
	These most well-known scaffold molecules are today undergoing clinical trials for which specifically interact with this viral target CA21-SP1-NC inside the cells might be
	targeting various types of tumor antigens for example, DARPin targeting VEGF-A (phase possible to inhibit or interfere with the normal HIV-1 replication cycle.
	II/III), VEGF-A/PDGF-B (preclinical), and VEGF/HGF (phase I) as well as another type 1.
	of protein binder, affibodies which specific to HER2 is now in phase I (11).
	Recently, it was established that a new type of artificial protein derived from the repeat
	protein family called Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A
	(PP2A), and the yeast kinase TOR1 (HEAT repeat protein) has a potential advantage in
	comparison to other well-known scaffold proteins (12). The HEAT repeat is a tandem
	repeat protein with a structural motif composed of two alpha helices linked by a short
	loop. It can form alpha solenoids which are found in a number of cytoplasmic proteins,
	often involved in intracellular transport and protein-protein interactions (13). Various
	repeat protein binders can be isolated by powerful bio-panning methods such as phage
	display or ribosome display. Many repeat proteins have been isolated and characterized,
	and one good example is GFP/EGFP-binding αReps (14). This repeat protein belonged
	to a new artificial repeat protein family in which their design is based on the thermostable
	HEAT-like repeats scaffold. They were able to colocalize with EGFP inside the different
	cell compartments without forming any aggregates or toxicity to the cells. Therefore, the
	αRep proteins are molecules of choice for the study of intracellular processes in living
	cells, by their direct interactions with endogenous protein of interest. In the present study
	we would like to apply αRep proteins to the development of a novel type of intracellular
	anti-HIV-1 treatment. The viral protein that we have targeted is a portion of the Pr55Gag
	polyprotein precursor overlapping the last 21 amino acids of the capsid (CA), the spacer
	peptide 1 (SP1), and the nucleocapsid domain (NC) which refers to CA21-SP1-NC. Many
	evidences showed that the C-terminal domain of CA (CACTD) is critical for the formation
	of viral capsid core and Gag oligomerization/polymerization in the step of viral assembly.

  integrase nuclear strand transfer inhibitors (INSTIs) also known as integrase inhibitors (INs) are the inhibitor that inhibit the function of viral enzyme integrase to prevent the integration of viral DNA into the DNA of the infected cell. There are several integrase inhibitors currently under clinical trial. Raltegravir is the one that became the first to receive FDA approval in 2007. It contains two metal binding groups that compete for substrate with two Mg 2+ ions at the metal binding site of integrase. Elvitegravir and dolutegravir are two other integrase inhibitors which already approved in early 2014 (65).

	14.) Protease inhibitors
	Protease inhibitors is a type of HIV-1 inhibitor which block the viral protease
	enzyme and prevent the production of mature virions upon budding from the
	host membrane. These drugs particulally block the cleavage of gag and

gag/pol precursor proteins.

[START_REF] Gilbert | Comparison of HIV-1 and HIV-2 infectivity from a prospective cohort study in Senegal[END_REF]

Virus particles produced in the presence of protease inhibitors are defective and mostly non-infectious. Examples of HIV protease inhibitors are lopinavir, indinavir, nelfinavir, amprenavir and ritonavir. Darunavir and atazanavir are currently recommended as first line therapy choices

[START_REF] Wensing | Fifteen years of HIV Protease Inhibitors: raising the barrier to resistance[END_REF]

.

LIST OF TABLES Page

Table 1 HIV-1 proviral integration in SupT1 cells harvested at day 14 pi. 103

109-139 from αRep-n4-a protein (light gray, PDB code 3LTJ, this work) were overlaid.

Positions D15, V20 and R21 in the αRep motif are superimposable on positions D17, V24

and R25 in the AAA-HEAT motif, as indicated by the arrow. The conserved proline P9 in the AAA-HEAT motif is not structurally equivalent to P8 in the αRep motif; these two (A) pQE-31-αRep4E3 and (B) pQE-31-αRep9A8

Electron microscopy analysis of HIV-1 Gag assembly into VLPs in

Sf/(Myr+)αRep4E3GFP or Sf/(Myr+)αRep9A8GFP

Recombinant baculovirus AcMNPV gag infected Sf9 cell control, Sf/(Myr+)αRep4E3-GFP and Sf/(Myr+)αRep9A8-GFP were harvested at 48 hours post-infection. Infected cells were pelleted and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.5, post fixed with osmium tetroxide (2% in H2O) and treated with 0.5% tannic acid solution in H2O. The samples were dehydrated and embedded in Epon (Epon-812, Fulham, Latham, NY). Ultrathin sections were stained with 2.6% alkaline lead citrate and 0.5% uranyl acetate in 50% ethanol, then post stained with 0.5% uranyl acetate solution in H2O. Grids were examined under a Jeol JEM-1400 electron microscope, equipped with an ORIUS TM digitalized camera (Gatan France, 78113-Grandchamp).

Construction of lentiviral vectors

The third generation of self-inactivating (SIN) CGW lentiviral vector (kindly provided by Dr. Bruce E. Torbett, The Scripps Research Institute, CA, USA) was used to prepare a backbone vector for transferring of the αRep protein coding genes; αRep4E3-EGFP and αRep9A8-EGFP into target cells. The vector contains MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with the myeoloproliferative sarcoma virus enhancer) to ensure the high level expression of transgenes. All constructs were generated by polymerase chain reaction (PCR) using pCEP4 which harboring each of αRep genes as a template. The PCR products were purified using GeneJET PCR Purification kit (Thermo Fisher Scientific, Waltham, MA) followed by treated with EcoRI (Thermo Fisher Scientific, Waltham, MA) and BstXI (New England Biolabs, Ipswich, MA). The insert genes were ligated into CGW lentiviral vector pretreated with the same restriction enzymes as shown in Figure 2.3 using T4 DNA ligase, then transformed the ligation products into the competent E. coli XL-1 Blue cells.

Transformation reactions were plated on LB agar containing 100 μg/ml of ampicillin. The single colonies were picked and preliminary identified by colony PCR and restriction enzymes analysis. The positive clones were then confirmed by DNA sequencing.

Flow cytometry

Flow cytometry was used to measure the efficacy of transfection or lentiviral vectors and HIV-1 virus transduction. Cell samples were harvested at 48 hours post-transfection or transduction. Cells were washed with PBS for the first round follow by 1% FBS-0.02% NaN3 in PBS. After washing step, the cells were resuspended in 1% paraformaldehyde in PBS and analyzed by flow cytometry. The samples were analyzed by flow cytometry (BD Accuri TM C6, BD Biosciences).

Confocal microscopy

To Excitation wavelengths were 405 nm for DAPI, 488 nm for EGFP and 561 nm for Qdot705. All renderings were processed using the Nikon NIS-elements of the AR 4.20.00 software.

Determination of p24 level by p24 antigen ELISA

The p24 level in culture supernatant collected from infected SupT1 cell control and SupT1 cell lines were quantified by p24 antigen ELISA (Bio-Rad, Marnes-la-Coquette, France) following the manufacturer's recommended protocol. The concentration of p24 from were calculated from the standard HIV-1 p24 antigen which was diluted to prepare a series of varying concentrations. A standard curve was constructed from which optical density values of culture supernatant samples are interpolated to determine their concentration.

Quantitation of integrated proviral DNA by SYBR RT-PCR-based integration assay

The integration of HIV-1 DNA in SupT1 stable cell lines and SupT1 control were quantified by using SYBR RT-PCR-based integration assay. The genomic DNA of infected SupT1 cell control and SupT1 stable cell lines were extracted by using the High Pure PCR template kit (Roche, Mannheim, Germany). The PCR reaction was performed by SensiFAST™ SYBR No-ROX Kit (Bioline, Luckenwalde, Germany) in total volume 20 μl as the standard protocol directed. The genomic DNA template used in the PCR was adjusted to 5 ng/μl and 1 μl was used per reaction. The reactions were performed using the CFX96 real-time PCR system with the following program: 20 second of hot start at 95°C, followed by 40 cycles of denaturation at 95°C for 30 second, annealing and extension at 68°C for 30 second.

Infectivity Assay of HIV-NL4-3 derived from SupT1 cells lines stably expressing αRep proteins

After quantification of the level of p24, the culture supernatant samples were adjusted to have equal amount of p24 at 15 μg/ml in fresh C-RPMI. Jurkat-GFP cells (105, 106) were prepared at 2. showed that we are able to observe the EGFP reporter protein in HeLa cells which transfected with our two αReps (Figure 3.10). Suggesting that αReps were produced by HeLa cell due to the construction of vector which were designed to fuse αRep genes to the EGFP gene at the upstream position.

HIV-1 proviral DNA detection

The level of viral genome integration in HIV-1-infected SupT1 cells and in SupT1 expressing αRep proteins was evaluated at day 14 pi by SYBR based-quantitative PCR amplification of host cell DNA extracts, using primers specific to the pol gene.

As shown in Table 1, there was no significant difference between αRep-expressing SupT1 cells and control cells, indicating that the αRep-mediated antiviral effect occured at the post-integration phase of the virus life cycle.

Table 1. HIV-1 proviral integration in SupT1 cells harvested at day 14 pi.

Figures in the Table represent the Cts values, mean ± SD (n=3).

Status of the extracellular HIV-1 genomic RNA molecules

CAp24-ELISA is routinely used to evaluate the quantities of HIV-1 particles released in the external milieu, since the amounts of non-particulate, free soluble CAp24 protein is negligible in the case of massive infection and lysis of infected cells. In order to confirm the negative effects of αRep proteins on HIV-1 particle formation and release, viral loads were assayed in the culture supernatants of αRep4E3-EGFP-and αRep9A8-EGFPexpressing SupT1 cells. As shown in Figure 3.13 A, the concentration of viral genome copies at D14 pi was significantly inferior in the culture media of SupT1/αRep4E3-EGFP and SupT1/αRep9A8-EGFP, compared to control samples, a result which correlated with the CAp24 levels. The results indicated that in SupT1/αRep4E3-EGFP, the concentration of genomic RNA copies was 8.7-fold lower than the control SupT1 cells, and 7.4-fold lower than the irrelevant αRep-expressing SupT1/αRep9C2-EGFP cells. With SupT1αRep9A8 cells, the concentration of genomic RNA copies was only 2.6-fold lower than the control SupT1 cells, and 2.2-fold lower than the SupT1/αRep9C2-EGFP cells (Figure 3.13 B). These results indicated that αRep4E3 had a more pronounced negative effect on genome packaging, compared to αRep9A8. Of note, D14 pi was therefore the point at which the inhibitory effect of αRep4E3 and αRep9A8 on the HIV-1 yields was the most pronounced, whereas at D21 the virus production resumed at D21 for the reasons of cell viability evoked above. To further confirm the negative effect on viral genome packaging of αRep proteins, the genome encapsidation was evaluated again by calculating the ratio of the concentration of extracellular viral genome copies to that of CAp24 antigen. Viral load assay were performed after normalized the CAp24 levels from all samples to be equal 25 μg and its efficiency was compared in the different SupT1 cell lines. The result confirmed that this inhibitory function was altered in both cell lines SupT1/αRep4E3-EGFP and SupT1/αRep9A8-EGFP, but to significantly different levels as showed in Figure 3.14.

The stronger negative effect observed in αRep4E3-expressing cells is 10-to 13-fold lower compared with SupT1 control cells and αRep9C2-expressing cells respectively, and 3-fold compared to αRep9A8-expreessing cells. This implied that the antiviral activity associated with αRep4E3 was predominantly directed against viral genome packaging.

Molecular mechanisms of the antiviral functions of αRep4E3 and αRep9A8

Two major parameters control the HIV-1 infectivity: (i) the degree of protease-mediated maturation of Pr55Gag, and more particularly the cleavage of CAp25 into CAp24 + SP1;

(ii) the genome content of the virus particles, and thus the ability of the viral particle to pack its genomic RNA during its morphogenesis process. Retroviruses in general produce large numbers of defective, genome-lacking particles, and their infectivity index (infectivity index = the ratio of infectious to noninfectious particles) is usually over 1:1,000. These two parameters were investigated in HIV-1-infected SupT1 cells expressing our different types of αRep proteins. The results obtained on D10 post-reinfection (Figure 3.17) indicated that the amounts of HIV-1 which egressed from SupT1/αRep4E3 (1.4%) and SupT1/αRep9A8 (1.25%) were significantly lower (15 to 17-fold) than those from control cells, SupT1 (24%) and SupT1/αRep9C2 cells (19.5%). At D10 postreinfection however, no difference was detectable in the infectious titers of the cell culture supernatants between SupT1/αRep4E3 and SupT1/αRep9A8. The Jurkat-GFP cultures were therefore further maintained, and cells analyzed by flow cytometry at D14 post-reinfection (Figure 3.17 In the present study, we designed another type of HIV-targeted scaffolds called αRep, which are based on a natural family of modular proteins constituted of alpha-helical HEAT repeats [START_REF] Urvoas | Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (alphaRep) based on thermostable HEAT-like repeats[END_REF]. Our viral target for the αRep screen, abbreviated CA21-SP1-NC, consisted of the carboxy terminal portion of the Gag polyprotein comprising of the fulllength NC protein with its two ZFs, the SP1 domain (14 residues) and the last 21 residues of the CA C-terminal domain (L343-L363). CA21-SP1-NC served as the viral bait in a phage display screen of our αRep protein library. We isolated two αRep proteins with a high affinity for CA21-SP1-NC, called αRep4E3 32 (kDa) and αRep9A8 (28kDa) by phage display technique. αRep4E3 contains 7 repeat motifs and αRep9A8 consists of 6 repeat modules. These two αRep proteins also showed the high binding efficacy to their target CA21-SP1-NC in vitro. The first antiviral effect of αRep4E3 and αRep9A8 were observed from the experimental designed to study the effect of αRep proteins on viral like particle (VLP) assembly in an insect cell expression system [START_REF] Gonzalez | Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay[END_REF]. We showed the quantitative effects of αRep4E3 and αRep9A8 on the amount of VLPs production in both western blotting and luciferase-based VLP production assay, and using a qualitative assay by electron microscopy. Sf9 cells stable expressing αRep4E3-GFP and αRep9A8-GFP mediated the negative effects to the VLPs production. Particles release from these two cell types displayed aberrant particle formation. Oval, irregular, bi-, triple-, or multiparticles budded in the same membrane envelope were mostly observed from the αRep4E3 expressing cells. It is possibly that interaction of αRep4E3 to CA21-SP1-NC may interfere Gag-Gag interaction, Gag oligomerization, and/or Gag-polymerization. particles as we observed the αRep4E3-GFP and αRep9A8-GFP at the same fractions of Gag protein in a dose dependent manner.

Consequently, after we confirmed the inhibitory effects of the two αRep proteins on the viral like particle formation, the next study was to test the antiviral effects against the laboratory virus strain, HIV-1NL4-3. SupT1 stable cell lines expressing αRep4E3-EGFP and αRep9A8-EGFP were constructed using SIN CGW third generation lentiviral vector as vehicle for gene delivery system. SupT1 cells expressing each of the αRep proteins were challenged with the HIV-1NL4-3 at 1 MOI. Viral replication inhibition were observed from both αRep4E3 and αRep9A8 as presented by the level of CAp24 antigen in the culture supernatant of infected SupT1 stable line cells as same as the level of genomic RNA copies. The phase of action of αRep4E3 and αRep9A8 took place at the late step of replication cycle as the SYBR RT-PCR based integration assay presented the same integration level in all αRep4E3, αRep9A8, SupT1 control, and αRep9C2 irrelevant αRep control. These data are assayed at D14 pi, the day that we can clearly see the negative effects of αRep4E3 and αRep9A8. D7 pi was not the suitable day for investigating the function of αRep protein because at the beginning or early infection virus does not cause effect much on cellular homeostasis. The level of CAp24 and viral genome copies in culture supernatant are less as well as the cells morphology, there is no cytopathic effect observed on this day except one giant cell in many field of 100x magnification.

Meanwhile, D21 pi seems to be too late to interpret the results on this day. Since the control set which included SupT1 control and αRep9C2 irrelevant αRep control were faced with the heavy infection according to they do not contain any antiviral scaffold molecule and became giant cells consequently lysis. By the time of subculture, new fresh media added into the well will dilute the level of CAp24 in the culture supernatant.

Although αRep4E3 and αRep9A8 were able to inhibit the virus replication at the same level as measured using CAp24, the level of viral genome copies were 3.4-fold difference.

This implied that the molecular mechanism of the two αRep proteins are not the same.

The results obtained from VLPs assembly assay confirm this hypothesis. The late phase of HIV-1 life cycle, can be subdivided into many phases including; (i) particle assembly, (ii) particle budding, (iii) particle maturation and infectivity. The αRep4E3 and αRep9A8 may be able to inhibit or negatively interfere one or more of these phases during the virus Mixed and stored at 4 ºC.

2.5 M CaCl2

CaCl2 (Mw. 111 g) 13.88 gm ddH20 30 ml

Stirred until dissolved and adjusted volume into 50 ml.

Filtered through 0.2 μm Millipore membrane filter.

Mixed and stored at -20 ºC.

0.1% TE buffer

TE buffer 100 μl

Added ddH20 to 100 ml Filtered through 0.2 μm Millipore membrane filter.

Mixed and stored at 4 ºC.