Simon Lachambre

Christophe Chopard

Bruno Beaumelle
email: bruno.beaumelle@cpbs.cnrs.fr

Preliminary characterization of nanotubes connecting T-cells and of their use by HIV-1

Background information. Cells, especially those of the immune system, can form long and thin connections termed tunneling nanotubes (TNTs). These structures can reach >100 µm in length and, in T-cells, contain actin but no tubulin and are not open ended. T-cell TNTs were found to form following cell contact and to enable the transfer of HIV-1 from an infected-to a connected-T-cell.

TNTs are poorly characterized at molecular level.

Results. We found Rab11 and tetraspanins, especially CD81, all along T-cells TNTs while Rab4 and Rab35 were absent from these structures. Regarding actin cytoskeleton regulators, Exo70, N-WASP and especially ezrin accumulated at the level of the TNT tip that contacts the connected cell.

Phosphoinositides such as PI(4,5)P2 were also concentrated at this level together with HIV-1 Gag.

Gag spots on cells and TNTs were essentially immobile, and likely correspond to area of Gag multimerisation for budding to form virus-like particles. Mobility of PHPLC a specific probe for PI(4,5)P2 was reduced > 3-fold at the level of TNT basis or tip compared to the cell body.

Conclusion.

Our study identified the TNT tip as an active zone of actin cytoskeleton reorganisation with the presence of ezrin, Exo70, N-WASP and PI(4,5)P2. The latter is also known to enable HIV-1 Gag recruitment for viral budding, and the presence of Gag at this level, contacting the connected cell indicates that the TNT tip is also a favourite place for HIV-1 assembly and budding.

Introduction

Cell contacts within the immune system are not restricted to neighbour cells and, during the recent years, evidence for long distance connections between distant cells accumulated. These thin structures have lengths of up to several cell diameters and were termed tunelling nanotubes (TNTs). They are longer than filopodia that are smaller than cell diameter [START_REF] Sherer | Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission[END_REF], and were first described in PC12 cells. In this cell type, TNTs have a diameter of 50-200 nm and contain actin but no microtubules [START_REF] Rustom | Nanotubular highways for intercellular organelle transport[END_REF]. TNTs were observed between various immune cells, such as B-cells and macrophages; TNTs of up to 140 µm in length were observed in this pioneer study [START_REF] Onfelt | Cutting edge: Membrane nanotubes connect immune cells[END_REF]. As reviewed recently [START_REF] Abounit | Wiring through tunneling nanotubes--from electrical signals to organelle transfer[END_REF], TNTs are not all identical. For instance, macrophages can produce two types of TNTs, thin ones (~0.3 µm in diameter) containing F-actin only and thick ones (~ 0.8 µm in diameter) that contain both F-actin and tubulin. The role of these different TNTs appears different since only small ones enabled bacteria to "surf" at their surface before phagocytosis by the destination cell, while only thick ones allowed the transfer of organelles such as mitochondria, late endosomes or lysosomes. TNTs were observed between macrophages whether they were plated on fibronectin or not [START_REF] Onfelt | Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria[END_REF]. This was not the case for T-cells that require adsorption onto fibronectin-coated substratum to produce TNTs. T-cells TNTs were found to contain actin but no tubulin and do not form open-ended tunnels since no intercellular transfer of cytosolic GFP or calcium flux was observed through these connections. Membrane proteins such as GPI-GFP or type I membrane proteins were not transferred either, indicating the absence of membrane continuity between the connected cells. No specific structure was observed by transmission electron microscopy at the site where the two parts of the TNTs interact [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF].

There is evidence for the existence of TNTs in vivo, between dendritic cells in the cornea for instance [START_REF] Chinnery | Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea[END_REF]. This type of membrane extensions also plays a crucial role during Drosophila development [START_REF] De Joussineau | Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila[END_REF]. Moreover, TNTs are exploited by several pathogens such as HIV-1. The use of humanized mice and multiphoton intravital microscopy showed that HIV-1 infected T-cells form TNT-like structures in vivo, and that HIV-1 uses these cell extensions to disseminate within lymph nodes [START_REF] Murooka | HIV-infected T cells are migratory vehicles for viral dissemination[END_REF]. Such HIV-1 transfer from an infected T-cell to an uninfected one via TNTs was also observed in vitro, using conventional cell culture [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]. Since, as indicated above, T-cells TNTs do not allow the direct transfer of membrane protein from one cell to the other, infection involves a cell-free (although presumably attached) intermediate. Indeed, a fusion inhibitor or neutralizing antibodies against gp120 or CD4 (respectively the viral glycoprotein and its receptor) prevented transmission [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]. It should also be noted that T-cell infection by HIV-1 did not induce TNT formation and that preventing HIV-1 transfer did not affect TNT number either [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF], indicating that TNTs are different from filopodia that are induced by HIV-1 Nef [START_REF] Nobile | HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes[END_REF], or those stabilized by Env that fall apart in the presence of neutralizing anti-Env antibodies [START_REF] Sherer | Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission[END_REF].

Despite their recognized role in HIV-1 transmission between T-cells, TNTs are poorly characterized at the molecular level. For instance it is not known whether cell membrane molecules involved in HIV-1 budding are present in TNTs. The first step in HIV-1 virion formation is the recruitment to the plasma membrane of the major HIV-1 structural protein, Gag. Expression of this single viral protein is sufficient to drive the assembly of virus-like particles (VLPs) that are morphologically similar to HIV-1 immature virions [START_REF] Fuller | Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle[END_REF]. The most recent data showed that VLP budding involves the same machinery as the one used by the entire virus [START_REF] Van Engelenburg | Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits[END_REF]. This key step of HIV-1 infection cycle has been recently reviewed [START_REF] Bell | HIV Gag polyprotein: processing and early viral particle assembly[END_REF]). An essential player to insure Gag targeting to the plasma membrane is phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). This phosphoinositide is specifically associated with the inner leaflet of the plasma membrane where it assembles in microdomains of ~73 nm in size as determined using super-resolution microscopy (van den [START_REF] Van Den Bogaart | Membrane protein sequestering by ionic protein-lipid interactions[END_REF]. Tetraspanins are a family of four transmembrane domain-containing proteins. They interact laterally with each other to form tetraspanin webs, also termed tetraspanin-enriched microdomains [START_REF] Espenel | Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web[END_REF] that were also found to colocalize with assembling Gag at the plasma membrane [START_REF] Ono | Relationships between plasma membrane microdomains and HIV-1 assembly[END_REF]. Moreover tetraspanins, especially CD81, negatively regulate HIV-1 cell to cell transmission but not cell-free virion infectivity [START_REF] Krementsov | Tetraspanins regulate cell-to-cell transmission of HIV-1[END_REF].

Here we found that PI(4,5)P2 and tetraspanins are present within TNTs, but with different localization. PI(4,5)P2 was found with Gag at the level of the TNT tip that establishes contact with the connected cell, while tetraspanins were observed all along the TNTs.

Results

Specificity of TNT imaging using fluorescence microsocopy

Throughout these studies we used Jurkat cells that were plated onto fibronectin coated coverslips before overnight incubation and either live-imaging or fixation with paraformadehyde with or without glutaraldehyde [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF]. We did not find that glutaraldehyde addition improved TNT preservation as long as paraformaldehyde was added at low concentration (0.1%) during cell washes before conventional fixation with 3.7% paraformaldehyde. Live imaging of T-cell TNTs is difficult because these tubes connect essentially spheric cells by their equatorial plane and therefore nanotubes do not lay on the substrate. They thus move, are fragile and sensitive to light exposure [START_REF] Abounit | Wiring through tunneling nanotubes--from electrical signals to organelle transfer[END_REF]. Indeed, strong illumination such as those needed for FRAP for instance can induce TNT movements.

Although previous reports indicated that 13% of Jurkat cells were connected by TNTs (Sowinski, et al., 2008) we never found more than 1/10 000 of Jurkat cells with TNTs and therefore could not reliably quantify them. Hence, although we found several cell molecules concentrated within TNTs, this lack of quantitative assay prevented us from studying their role in TNT formation, by using siRNAs for instance. Throughout this study we avoid imaging as TNT membranous tethers that connect daughter cells at the end of cell division, before abscission. These connections can be identified by the presence of a characteristic midbody that causes a bulge in the middle of the tube [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]. Finally, please note that because TNTs are thin structures that contain a limited amount of fluorophores, their visualization usually requires acquisition settings such as the signal from the cell body is saturated.

Absence of lipophilic dye transfer through nanotubes

Jurkat T-cells were labelled with a lipophilic dye (DiI or DiD [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF]) before mixing the two populations, plating onto fibronectin-coated coverslips, overnight incubation, fixation and observation by confocal microscopy. As observed before [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF], T-cells can form two types of TNTs. We found that most of them are extensions from a single cell only while others are made of extensions from the two connected cells. Lipophilic tracers were not transferred from one cell to the other via TNTs indicating that membrane discontinuity is preserved at the junction between connected cells (Fig. 1A).

Actin, ezrin and N-WASP are present in TNTs

As reported earlier [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF], T-cell TNTs contain actin (Fig. 1F) but few  tubulin (Fig. 1B), indicating that these structures are devoid of microtubules. We then examined whether regulators of actin polymerisation were present within TNTs. Cdc42 and Rac GTPases are key regulators of membrane extension such as lamellipodia and filopodia [START_REF] Heasman | Mammalian Rho GTPases: new insights into their functions from in vivo studies[END_REF], and we found them at the basis of TNTs only, close to the cell body of the transfected cell (Fig. 1E andF). N-WASP is involved in connections between the membrane and the cytoskeleton and has been shown to localize to certain types of filopodia [START_REF] Takenawa | The WASP-WAVE protein network: connecting the membrane to the cytoskeleton[END_REF]. We found it within TNTs (Fig. 1D), not only at the basis but also at the tip of the TNT, indicating that active actin polymerization takes place at these levels. We also studied the localization within TNTs of ezrin, a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are involved in membrane-cytoskeleton interaction and in the organization of membrane domains [START_REF] Fehon | Organizing the cell cortex: the role of ERM proteins[END_REF]. T-cells express no radixin, traces of ezrin and a lot of moesin [START_REF] Faure | ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation[END_REF]. We found that ezrin-GFP, a chimera validated earlier [START_REF] Lamb | Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts[END_REF], accumulated at the basis and, interestingly, at the tip of the TNT (Fig. 1C). PI(4,5)P2 and PI(3,4,5)P3 are enriched at the tip of the TNT Because ezrin is activated by phosphorylation and binding to PI(4,5)P2 [START_REF] Fehon | Organizing the cell cortex: the role of ERM proteins[END_REF] we examined, using a GFP chimera of the PH domain of PLC that specifically binds this phosphoinositide [START_REF] Varnai | Visualization of phosphoinositides that bind pleckstrin homology domains: calcium-and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools[END_REF], whether it was present within TNTs. We found that PI(4,5)P2 is specifically enriched in the distal part of the TNT at the site of junction with the contacted cell (Fig. 2), i.e. where ezrin accumulated (Fig. 1C). The other phosphoinositide PI(3,4,5)P3, localized with the Akt PH domain [START_REF] Astoul | PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models[END_REF], was also present at the TNT tip (Fig. 2). The specific enrichment of these probes at the TNT tip was observed in 65-70% of the TNTs we observed. It should be noted that this value is likely an underestimate because some TNT tips were either not in the confocal section or difficult to identify due to signal saturation at the target cell periphery.

Cytoplasmic proteins have access to TNT

As observed before [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF], cytoplasmic proteins such as GFP or a GFP chimera of the R40L mutant of PHPLC that is unable to bind PI(4,5)P2 [START_REF] Varnai | Visualization of phosphoinositides that bind pleckstrin homology domains: calcium-and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools[END_REF] were found within TNTs (Fig. 3). The fact that the PHPLC mutant was present throughout the TNT length (Fig. 3) shows that the specific accumulation of WT PHPLC at the TNT tip (Fig. 2) is due to its capacity to bind PI(4,5)P2.

Are membrane-deforming proteins present within TNTs?

We then studied the localization in TNTs of proteins known to deform membrane and induce filopodia. Missing-in-metastasis (MIM) is a member of the BAR (Bin, Amphiphysin, Rvs) domain superfamily of proteins. MIM is an actin-binding protein that directly binds PI(4,5)P2 and deforms membrane by inducing membrane protrusions. This activity is due to the presence of an I-BAR domain at its N-terminus [START_REF] Zhao | I-BAR domain proteins: linking actin and plasma membrane dynamics[END_REF]. As observed in other cell types [START_REF] Zhao | I-BAR domain proteins: linking actin and plasma membrane dynamics[END_REF], expression of a GFP chimera of MIM I-BAR domain induced dramatic changes in T-cell shape, with the generation of several filopodia (Fig. 4). Most often, these elongations did not contact other cells and are therefore different from bona fide TNTs. This result suggests that MIM is not responsible for TNT generation, although it does not rule out that MIM could be present in TNTs. Exo70 is another protein able to induce filopodia upon overexpression within cells [START_REF] Zhao | Exo70 generates membrane curvature for morphogenesis and cell migration[END_REF]. Its transient expression in T-cells did not lead to the formation of filopodia although exo70 was observed within TNTs (Fig. 4). T-cells are largely devoid of M-Sec [START_REF] Ohno | M-Sec: Emerging secrets of tunneling nanotube formation[END_REF], nevertheless transient expression of M-Sec in macrophages induces the formation of numerous membrane protrusions that connect cells, contain actin but not microtubules and allow calcium flux [START_REF] Hase | M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex[END_REF]. We found that transfected GFP-M-Sec did not localize to TNTs and did not induce cell protrusions in T-cells (Fig. 4). This result is consistent with previous observations indicating that TNTs are different structures in macrophages [START_REF] Hase | M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex[END_REF] and T-cells [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF]. Hence, of the three proteins able to induce membrane curvature that we tested, only Exo70 seems to localize to bona fide TNTs. This result is consistent with the capacity of PI(4,5)P2 to recruit Exo70 to the plasma membrane [START_REF] Liu | Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells[END_REF]. Moreover, Exo70 is known to stimulate the Arp2/3 complex to promote actin filament nucleation and branching [START_REF] Liu | Exo70 stimulates the Arp2/3 complex for lamellipodia formation and directional cell migration[END_REF]. Its localization to the TNT tip confirms that active actin polymerisation likely takes place at this level.

T-cells TNTs are essentially devoid of elements of the exocytic and endocytic pathway

It is still unclear whether T-cells TNTs contain elements of the exocytic or endocytic pathway. To examine this point we first transiently expressed YFP-KDEL [START_REF] Bannai | Kinesin dependent, rapid, bi-directional transport of ER sub-compartment in dendrites of hippocampal neurons[END_REF]. We did not find significant amount of this ER tracer within TNTs (Fig. 5). Regarding the endocytosis pathway we used different Rab proteins as markers [START_REF] Mitra | Rab GTPases implicated in inherited and acquired disorders[END_REF]. In Jurkat cells, Rab4 is concentrated within the perinuclear recycling compartment. Rab11 is observed within these recycling endosomes, but also to some extend at the plasma membrane. Rab5 is associated with endosomal compartments dispersed throughout the cytoplasm, while Rab35 essentially localized to the plasma membrane (SupFig.1). We observed Rab4 and Rab35 at a low level within TNTs (Fig. 5). Similar data were obtained for Rab5 (data not shown). Rab11 was more concentrated in TNTs but showed a linear labelling pattern consistent with plasma membrane labelling and not accumulation within intra-TNT structures. Although it was somehow surprising to find that Rab11 is more concentrated than Rab35 within TNTs, these data suggest that endocytic traffic does not take place within TNTs. They were confirmed when fluorescent transferrin was used (see below). This interpretation is also consistent with electron microscopic examination of these cell extensions that did not show evidence for vesicular or cisternal structures within T-cell TNTs [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF].

T-cells TNTs are enriched in tetraspanins

Tetraspanins were good candidates to associate with TNT because the tetraspanin web is known to interact with ERM proteins [START_REF] Charrin | Lateral organization of membrane proteins: tetraspanins spin their web[END_REF][START_REF] Sala-Valdes | EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrinradixin-moesin proteins[END_REF] that we found in TNTs (Fig. 1C). Indeed we found that both CD9 and CD81 accumulated within TNTs. More specifically, CD81-mCherry was even more sensitive than phalloidin conjugates to insure sensitive visualization of these structures (Fig. 6). Although CD81 could be observed with PHPLC at the level of the TNT tip, tetraspanins did not show the specific accumulation at the TNT apex that was observed for ezrin (Fig. 1C) or PI(4,5)P2 (Fig. 2).

HIV-1 Gag accumulates at the TNT tip

TNTs were previously found to allow HIV transfer to the connected cell but the mechanistic of HIV-1 targeting to these structures was not studied [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF]. To examine this point we used a Gag-GFP chimera that can form VLPs just as native Gag [START_REF] Hermida-Matsumoto | Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging[END_REF]. At low expression level, Gag-GFP showed a diffuse cytosolic labelling with a few spots on the plasmalemma (Fig. 7A), while at higher expression level these spots became more numerous (Fig. 7B). When T-cells were cotransfected with CD81-mCherry and Gag-GFP we found that Gag localized to TNTs, as observed before with the complete virus [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF]. More specifically, we most often found Gag accumulation at the TNT tip, at the site of contact with the connected cell(s) (Fig. 7B), just where PI(4,5)P2 is present (Fig. 2). This observation is consistent with the crucial role of this phosphoinositide for Gag targeting to the plasma membrane and for HIV-1 budding [START_REF] Bell | HIV Gag polyprotein: processing and early viral particle assembly[END_REF]. Along the same lines, the partial colocalization of Gag with CD81 at the TNT tip (Fig. 7B, inset) is consistent with the CD81 colocalization with PHPLC at this level (Fig. 6).

Mobility of PI(4,5)P2 and HIV-1 Gag on TNTs

Does HIV-1 move along TNTs, by viral "surfing" for instance [START_REF] Lehmann | Actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells[END_REF] or is it assembled at different sites along these structures, especially at the tip? To examine this point we used fluorescence recovery after photobleaching (FRAP). As observed before in HeLa cells [START_REF] Helma | Direct and dynamic detection of HIV-1 in living cells[END_REF][START_REF] Jouvenet | Imaging the biogenesis of individual HIV-1 virions in live cells[END_REF], in most cases (>80%), we did not observe significant fluorescence recovery following bleaching of Gag-GFP spots at the plasma membrane (not shown) or TNTs of Jurkat cells (Fig. 8A). This result indicates that these spots are stable and probably correspond to VLP assembly sites. To assess whether this immobility was due to a restricted diffusion of PI(4,5)P2, we monitored its mobility using PHPLC-GFP. This probe was found to diffuse rapidly with an apparent diffusion coefficient (D) ~ 1.2 µm 2 / s at the plasma membrane of HEK cells [START_REF] Hammond | Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information[END_REF]. This value is similar to those obtained for a fluorescent analog of PI(4,5)P2 [START_REF] Golebiewska | Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells[END_REF][START_REF] Golebiewska | Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages[END_REF]. We accordingly observed D~1.1 µm 2 / s at the surface of Jurkat cells, and similar values were obtained in the middle of TNTs, indicating that membrane curvature did not significantly affect D measurements in this experimental system (Fig. 8B). Nevertheless, D was significantly decreased to D~0.3 µm 2 /s at the level of cell extensions or at the tip or basis of TNTs. Hence, although PI(4,5)P2 mobility was reduced at the level of TNT ends it remained significant and is not responsible for the immobility of Gag spots on these structures.

Discussion

Evidence is accumulating that TNTs play a major role in cell communication within elaborated organs [START_REF] Davis | Membrane nanotubes: dynamic long-distance connections between animal cells[END_REF]. In the case of T-cells, TNT-like structures have been shown to promote HIV-1 dissemination within lymph nodes in vivo [START_REF] Murooka | HIV-infected T cells are migratory vehicles for viral dissemination[END_REF]. This mode of viral transmission seems to be especially relevant for T-tropic viruses such as HIV-1 and HTLV-1 whose dissemination in the body essentially takes place via cell contacts [START_REF] Igakura | Spread of HTLV-I between lymphocytes by virus-induced polarization of the cytoskeleton[END_REF][START_REF] Martin-Serrano | Host factors involved in retroviral budding and release[END_REF]. Although TNTs were known to contain cytosolic proteins, actin but not tubulin [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF], little was known regarding the other structural elements that make up T-cell TNTs. Among the different markers we used for the endocytic or the exocytic pathway, none of them was significantly concentrated within TNTs, indicating that these cell extensions do not contain intermediates of the endocytic or exocytic pathways. We nevertheless found that Rab11 and tetraspanins, especially CD81, were present all along the TNT membrane. This localization was clearly different from that of PI(4,5)P2, ezrin and HIV-1 Gag that were enriched at the TNT apex, where contact with the connected cell takes place. The TNT tip thus seems to be a specific domain of this structure.

The significant reduction in PI(4,5)2 mobility within TNTs ends (i.e. basis and tip) could indicate the presence of a fence limiting PI(4,5)2diffusion as is the case at the level of the phagocytic cup [START_REF] Golebiewska | Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages[END_REF] or, more probably, that this phosphoinositide interacts with proteins at this level. Indeed, we found several PI(4,5)P2 binding proteins involved in actin cytoskeleton organization such as ezrin [START_REF] Fehon | Organizing the cell cortex: the role of ERM proteins[END_REF], N-WASP [START_REF] Thrasher | WASP: a key immunological multitasker[END_REF]) and exo70 [START_REF] Liu | Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells[END_REF] at the TNT tip. It is well established that areas of the plasma membrane enriched in PI(4,5)P2 correspond to places where active actincytoskeleton remodeling takes place [START_REF] Di Paolo | Phosphoinositides in cell regulation and membrane dynamics[END_REF].

The localisation of ezrin within TNTs agrees well with the data from a pioneer study that localized moesin, another ERM protein, to dynamic filopodia [START_REF] Amieva | Subcellular localization of moesin in dynamic filopodia, retraction fibers, and other structures involved in substrate exploration, attachment, and cell-cell contacts[END_REF]. Moreover, PI(4,5)P2 enables Gag targeting to the plasma membrane for HIV-1 budding [START_REF] Bell | HIV Gag polyprotein: processing and early viral particle assembly[END_REF][START_REF] Martin-Serrano | Host factors involved in retroviral budding and release[END_REF][START_REF] Ono | Relationships between plasma membrane microdomains and HIV-1 assembly[END_REF]. ERM proteins are incorporated within HIV-1 virions during budding, with ezrin and moesin present at a level of ~2 molecules /100 Gag [START_REF] Ott | Cytoskeletal proteins inside human immunodeficiency virus type 1 virions[END_REF]. It was therefore not surprising to find PI(4,5)P2, ezrin and Gag in the same place in TNTs. This observation strongly suggests that the TNT tip is a productive site for HIV-1 assembly to infect the connected cell, a kind of "mini" virological synapse. The fact that VLP assembly sites are immobile on TNTs suggests that Gag monomers diffuse through the TNT lumen, just as it is the case for cytosolic proteins, before being recruited by PI(4,5)P2 at the TNT tip for budding in proximity of the connected cell. Nevertheless, our data obtained using VLPs do not exclude that HIV-1 could also be transported on the TNT surface via the envelope protein gp120.

The partial colocalization of the tetraspanin CD81 with Gag we observed at the TNT tip (Fig. 7B, inset) is consistent with the reported interaction of CD81 with PI-4 kinase, a key enzyme in PI(4,5)P2 synthesis [START_REF] Berditchevski | A novel link between integrins, transmembrane-4 superfamily proteins (CD63 and CD81), and phosphatidylinositol 4-kinase[END_REF]. Moreover, the tetraspanin web interacts with ERM proteins [START_REF] Charrin | Lateral organization of membrane proteins: tetraspanins spin their web[END_REF][START_REF] Sala-Valdes | EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrinradixin-moesin proteins[END_REF] that bind PI(4,5)P2 [START_REF] Fehon | Organizing the cell cortex: the role of ERM proteins[END_REF]. Altogether these data indicate that the tetraspanin web is present in close proximity with PI(4,5)P2 membrane domains and might regulate HIV-1 budding. To pursue this study, because of the small TNT diameter (<200 nm [START_REF] Sowinski | Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission[END_REF]), it would be interesting to use a high resolution technique with a resolution <20-30 nm such as photoactivation localization microscopy for instance [START_REF] Toomre | A new wave of cellular imaging[END_REF].

Materials and Methods

Chemicals

DiD, DiI and fluorescent phalloidins were from Life Technologies. Human fibronectin and transferrin were from Sigma. Transferrin was labelled with Cy3 or Cy5 according to the instructions of the manufacturer (Life Technologies).

Plasmids

Mammalian expression vectors for the following constructs were obtained from the indicated individuals. YFP-Tubulin and YFP-KDEL were from Fabien Blanchet (CPBS, Montpellier), ezrin-GFP from Paul Mangeat, GFP-Cdc42 and GFP-Rac1 from Cécile Gauthier-Rouvière, GFP-Rab4 and GFP-Rab11 from Anne Blangy (CRBM, Montpellier), CD9-GFP and CD81-mCherry from Pierre-Emmanuel Milhiet (CBS, Montpellier), GFP-N-WASP from Pascale Cossart (Institut Pasteur, Paris), PH PLC R40L-GFP from Tamas Balla (NIH, Bethesda), GFP-Exo70 from Wei Guo (University of Pennsylvania, Philadelphia), GFP-M-Sec from Hiroshi Ohno (RIKEN, Kanagawa), MIM-I-Bar-GFP from Pekka Lappalainen (Institute of Biotechnology, Helsinki), GFP-Rab35 from Arnaud Echard (Institut Pasteur Paris). pEGFP was from Clontech and Gag-EGFP from the NIH AIDS reagent program. The GFP chimera of PHPLC and PHAkt were described before [START_REF] Rayne | Phosphatidylinositol-(4,5)bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF].

Cell transfection and TNT observation

Jurkat T-cells were used throughout this study. Cell labelling with DiD or DiI was performed as described [START_REF] Sowinski | Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1[END_REF]. Cells were transfected by electroporation [START_REF] Vendeville | HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses[END_REF]) before plating 0.8.10 6 cells in 1 ml of medium onto 18 mm-diameter coverslips in 12-well plates. Coverslips were previously coated with fibronectin (10 µg/ml PBS). After overnight incubation, cells were either imaged directly or washed gently twice with PBS containing 0.1% paraformaldehyde before fixation in 3.7 % paraformaldehyde for 2 h at RT. Following treatment with 50 mM ammonium chloride, cells were either mounted directly or permeabilized with saponin for labelling F-actin with phalloidin [START_REF] Rayne | Phosphatidylinositol-(4,5)bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. Samples were imaged using an SPE Leica confocal microscope with a 63x objective (NA 1.33). Confocal sections were centred on the TNT and were ~2 µm thick.

FRAP

Jurkat cells were transfected as described above and plated on fibronectin-coated 35 mm diameter glassbottom Ibidi dishes. After overnight incubation, the medium was changed to medium without phenol red and 10 mM Hepes was added before imaging at 37°C using a Zeiss LSM780 confocal microscope and a 63x (NA 1.4) objective. Bleached area were circles of diameter between 0.4 and 4 µm 2 , bleaching time was < 1.5 s, scanning times were < 100 ms and images were collected with an interval of ~0.2 s. 12 bits images were recorded. Calculations were performed using imageJ, using when needed a correction for scanning-induced bleaching that remained < 15% of initial fluorescence intensity. A simplified equation was used to obtain diffusion coefficients from confocal FRAP data [START_REF] Kang | Simplified equation to extract diffusion coefficients from confocal FRAP data[END_REF]. Experimental D values were not corrected for membrane curvature (see Results).

Author contribution

CC prepared plasmids and performed some transfection experiments. SL was in charge of cell imaging. BB did some imaging, directed the study and wrote the paper.

Figure 1 .FFigure 2 .

 12 Figure 1. Actin, ezrin and N-WASP are enriched in T-cell TNTs. (A) Jurkat T-cells were labelled with a lipophilic dye DiD (blue) or DiI (red). The two populations were then mixed (1/1), layered on fibronectin coated coverslips, fixed after overnight incubation and imaged by confocal microscopy. (B-F) Jurkat cells were transfected with (B) YFP-Tubulin and CD81-mcherry, (C) ezrin-GFP, (D) GFP-N-WASP, (E) GFP-Rac1 or (F) GFP-Cdc42 before overnight incubation, fixation and imaging. When indicated cells were permeabilized for F-actin staining with fluorescent phalloidin. Connected cell bodies are indicated by ellipses in some images that are representative of 20 (A), 6 (B), 11 (C), 13 (D), 13 (E) and 7 (F) nanotubes. Bars, 5 µm.

Figure 3 .

 3 Figure 3. Cytosolic proteins are present in T-cells TNTs. Jurkat cells were transfected with GFP or a GFP version of the R40L mutant of PHPLC (PHmut) that is cytosolic (Varnai and Balla, 1998), as indicated. Connected cell bodies are indicated by ellipses when needed for visualization. Images are representative of 6 (GFP) or 8 (PHmut) TNTs. Bars, 5 µm.

Figure 4 .

 4 Figure 4. TNTs contain Exo70. Jurkat cells were transfected with the GFP chimera of Exo70, mSec or MIM-I-Bar. When indicated, transfected cells were mixed with DiI labelled cells. Images are representative of 15 (Exo70), 6 (mSec) or 15 (MIM) TNTs. Bars, 5 µm.

Figure 5 .

 5 Figure 5. Endoplasmic reticulum and endocytic structures are not present within T-cells TNTs. Cells were transfected with YFP-KDEL or GFP-Rab proteins. When indicated, transfected cells were mixed (1/1) with DiD-labelled cells before adding onto fibronectin-coated coverslips. Images are representative of 6 (YFP-KDEL), 6 (Rab4) , 7 (Rab11) or 14 (Rab35) TNTs. Bars, 5 µm.

Figure 6 .

 6 Figure 6. Tetraspanins are enriched in T-cell TNTs. Cells were transfected with CD9-GFP or CD81-mCherry with or without PHPLC-GFP as indicated, before overnight incubation on fibronectin-coated coverslips. When indicated, F-actin was stained with fluorescent phalloidin. The connected cell periphery is indicated by a dotted line when needed. Images are representative of 15 (CD9/actin), 17(CD81/actin) or 16 (CD81/ PHPLC) TNTs. Bars, 5 µm.

Figure 7 .Figure 8 .

 78 Figure 7. HIV-1 virus like particles assemble at the TNT tip. Jurkat cells were transfected with Gag-GFP without (A) or with (B) CD81-mcherry and incubated overnight onto fibronectin-coated coverslips. When indicated (A), 100 nM fluorescent transferrin was added 40 min before fixation. Transferrin is absent from TNTs. When needed (B) the connected cell periphery is indicated by a dotted line. 3 out of 4 TNTs with tips within the confocal section show a Gag-GFP spot on the connected cell. The boxed region is enlarged in the lowest panels. Images are representative of 7 (A) or 20 (B) TNTs. Bars, 5 µm.

Acknowledgements

We acknowledge all the people that provided expression vectors for this study, Daniel Davis and Patrick Bron for discussions, Virginie Georget and Sylvain de Rossi for help with FRAP and live cell imaging, respectively, Pierre-Emmanuel Milhiet for carefully reading the manuscript and Cyril Favard for help with FRAP analysis. This work was funded by Sidaction and the CNRS through the PEPS program.


Malvina Schatz

Phuoc Bao

Viet Tong

Bruno Beaumelle
email: bruno.beaumelle@irim.cnrs.fr

Unconventional secretion of viral proteins

Although largely less numerous and characterized than bacterial secreted effectors, several viral virulence factors are secreted by virus infected cells. However, their mode of secretion only starts to be studied at the molecular level. Several of these viral effectors are secreted using an unconventional secretion pathway, i.e. despite the lack of signal sequence. We here review recent results illustrating the diversity of these pathways. In the case of HIV-1 proteins Tat and matrix (p17) proteins, secretion directly takes place at the plasma membrane level following binding to PI(4,5)P2. The secretion of HTLV-I Tax was found to partly rely on exocytic pathway intermediates. The secretion pathways of VP22 of Herpes simplex virus type I and VP40 of the Ebola virus are less well characterized but VP40 can be recruited to the plasma membrane by PI(4,5)P2 that thus appears as a key partner enabling the unconventional secretion of many viral proteins. Several studies indicated that circulating retroviral transactivating proteins Tat and Tax are involved in the development of AIDS and HTLV-I associated myelopathy/tropical spastic paraparesis, respectively. This review is part of a Special Issue of SCDB on "unconventional protein secretion" edited by Walter Nickel and Catherine Rabouille.

Introduction

Most viruses modify infected cells so that these pathogens can easily and safely multiply intracellularly. Accordingly, several viral effectors have an intracellular target that they will either activate or neutralize. For instance, HIV-1 Nef protein prevents MHC class-I molecules transport to the cell surface thereby preventing infected cell lysis by cytotoxic CD8 T-cells [START_REF] Geyer | Structure--function relationships in HIV-1 Nef[END_REF]. However, viral proteins are often multifaceted proteins and the role of some of them is not limited to their intracellular function. Infected cells indeed actively secrete some viral effectors, and some key virulence factors are exported in the absence of signal sequence. They therefore follow an unconventional secretion pathway. This is the case of retrovirus transactivating proteins Tat (HIV-1) [START_REF] Ensoli | Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients[END_REF] and Tax (HTLV-1) [START_REF] Alefantis | Secretion of the human T cell leukemia virus type I transactivator protein tax[END_REF], of Ebola virus VP40 matrix protein [START_REF] Reynard | Unconventional secretion of Ebola virus matrix protein VP40[END_REF] and of herpes virus VP22 [START_REF] Elliott | Intercellular trafficking and protein delivery by a herpesvirus structural protein[END_REF]. They therefore follow an unconventional protein secretion (UPS) pathway.

UPS pathways of signal peptide lacking proteins fall in three types. Type I UPS refers to cargo translocation across the plasma membrane, either through self translocation, or with the help of other protein complexes and pores. Type II refers to the use of ABC transporter, and Type III corresponds to secretion of cytoplasmic proteins that reach the lumen of a membrane-bound organelle or that are associated with a membrane-bound organelle during their export route. In addition, they can be released via extracellular vesicles, microvesicles shedding from the plasma membrane and/or by exosomes [START_REF] Rabouille | Pathways of Unconventional Protein Secretion[END_REF].

In this review we will present and discuss the evidences for the unconventional secretion of viral proteins, the role of these secreted proteins in viral multiplication and their possible association with the development of associated disease(s).

*HIV-1 Tat

HIV-1 transactivating protein (Tat) is a small (10 kDa, 86-102 residues) and basic protein that is crucial for viral transcription. Although essentially observed at the plasma membrane level in primary CD4+ T-cells (the main targets of the virus), Tat concentrates into the nucleus of most cell lines [START_REF] Rayne | HIV-1 Tat is unconventionally secreted through the plasma membrane[END_REF][START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. However, although devoid of signal peptide, Tat was detected in the extracellular medium in the absence of cell lysis [START_REF] Ensoli | Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients[END_REF]. Moreover, Tat secretion was not inhibited by brefeldin A [START_REF] Chang | HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region[END_REF], a drug that disrupts the Golgi and inhibits the ER to Golgi conventional secretion pathway [START_REF] Chardin | Brefeldin A: the advantage of being uncompetitive[END_REF]. Collectively, these observations indicated that Tat is secreted using an unconventional pathway. This secretion process was found to rely on Tat recruitment to the plasma membrane by phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) (Fig. 1) [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF], as it is the case for FGF2 secretion [START_REF] Temmerman | A direct role for phosphatidylinositol-4,5bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF].Tat has a very strong affinity for PI(4,5)P2, much stronger than that of cellular proteins. This is because to bind PI(4,5)P2 Tat not only uses the 49-51 basic-residue triplet within its basic domain to recognize PI(4,5)P2 head group, but also inserts the side chain of its single Trp (W11) in the membrane. In fact, Tat requires a lipid membrane to bind PI(4,5)P2. Hence, contrarily to FGF2 [START_REF] Temmerman | A direct role for phosphatidylinositol-4,5bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF], Tat does not recognize PI(4,5)P2 when spotted on nitrocellulose [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. The key role of Tat Trp in PI(4,5)P2 binding was confirmed using conservative mutations such as Tat-W11Y that strongly affected Tat binding to PI(4,5)P2. Trp side chain insertion into the membrane upon PI(4,5)P2 binding is a first and key step of Tat translocation to the trans-side of the membrane. Accordingly, Tat mutants unable to bind PI(4,5)P2 such as Tat-(49-51)A or Tat-W11Y failed to be secreted. Moreover, PI(4,5)P2 depletion using 5-phosphatases or PI(4,5)P2 masking using neomycin (a polycationic antibiotic) inhibited Tat secretion, thereby confirming the crucial role of PI(4,5)P2 in Tat unconventional secretion [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. Recently, as reported earlier for FGF2 [START_REF] Zacherl | A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2[END_REF], Tat and Tat-fusion proteins were found to bind to the alpha subunit of the Na + /K + ATPase. Accordingly, just as FGF2 [START_REF] Florkiewicz | The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na + ,K + -ATPase[END_REF], Tat secretion by HEK cells was inhibited by ouabain, a well-known inhibitor of the Na + /K + ATPase [START_REF] Agostini | Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection[END_REF]. Nevertheless, ouabain did not affect Tat secretion by T-cells [START_REF] Rayne | HIV-1 Tat is unconventionally secreted through the plasma membrane[END_REF] and the role of the Tat-Na + /K + ATPase interaction in Tat secretion is therefore unclear. Following PI(4,5)P2 binding, membrane insertion and translocation to the trans side of the membrane, Tat interacts with heparin sulfate proteoglycans (HSPG) that likely facilitate the refolding of secreted Tat [START_REF] Chang | HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region[END_REF], as it is the case for FGF2. Indeed, HSPGs are well known to bind extracellular Tat [START_REF] Tyagi | Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans[END_REF] and FGF2 [START_REF] Zehe | Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2[END_REF]. These interactions are most likely electrostatic driven since HSPGs are highly sulfated anionic polymers [START_REF] Sarrazin | Heparan sulfate proteoglycans[END_REF] , while Tat and FG2 are basic proteins with pIs of 10 and 11, respectively. Tat translocation step per se is poorly understood (Fig. 1) and it is still not clear whether it requires a cytosolic chaperone. It might need unfolding since, contrarily to VP22, Tat is unable to export galactosidase (a huge 476 kDa protein) from cells [START_REF] Mori | Efficient secretion of the herpes simplex virus tegument protein VP22 from living mammalian cells[END_REF]. An in vitro study using PI(4,5)P2 liposomes and purified Tat indicated that Tat can form pores into membranes [START_REF] Zeitler | HIV-Tat Protein Forms Phosphoinositidedependent Membrane Pores Implicated in Unconventional Protein Secretion[END_REF], as does FGF2 [START_REF] Muller | Formation of disulfide bridges drives oligomerization, membrane pore formation, and translocation of fibroblast growth factor 2 to cell surfaces[END_REF]. Liposome destabilization was followed by the release of encapsulated calcein and Tat mutants unable to bind PI(4,5)P2 were only weakly active in this [START_REF] Zeitler | HIV-Tat Protein Forms Phosphoinositidedependent Membrane Pores Implicated in Unconventional Protein Secretion[END_REF]. Whether Tat can form pores in the plasma membrane for secretion is not known yet. Several evidences indicate that extracellular, circulating Tat is involved in HIV-1 pathogenesis. First, Tat strongly increases the expression of HIV-1 co-receptors CXCR4 and CCR5 at the surface of CD4+ T-cells, thereby facilitating viral infection [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF][START_REF] Huigen | Multiple effects of HIV-1 trans-activator protein on the pathogenesis of HIV-1 infection[END_REF]. Extracellular Tat also triggers the production of immunoregulatory cytokines such as IL-10 by lymphocytes, monocytes and macrophages, thereby contributing to immune suppression [START_REF] Huigen | Multiple effects of HIV-1 trans-activator protein on the pathogenesis of HIV-1 infection[END_REF][START_REF] Rayne | The Ability of Chloroquine To Prevent Tat-Induced Cytokine Secretion by Monocytes Is Implicated in Its In Vivo Anti-Human Immunodeficiency Virus Type 1 Activity[END_REF]. Tat is also present in the central nervous system. It can either cross the blood-brain barrier or be produced in situ by infected microglial cells [START_REF] Bagashev | Roles and functions of HIV-1 Tat protein in the CNS: an overview[END_REF]. Tat was found to be toxic both for neurons [START_REF] New | Human immunodeficiency virus type 1 Tat protein induces death by apoptosis in primary human neuron cultures[END_REF] and astrocytes [START_REF] Eugenin | HIV-tat induces formation of an LRP-PSD-95-NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes[END_REF]. Tat also affects endothelial cells, thereby damaging the blood brain barrier [START_REF] Strazza | Breaking down the barrier: the effects of HIV-1 on the blood-brain barrier[END_REF]. Hence, Tat is suspected to be associated with the development HIV-1 associated neurological disorders which are among the most severe sources of morbidity for AIDS patients, even under highly active antiretroviral therapy [START_REF] Ghafouri | HIV-1 associated dementia: symptoms and causes[END_REF].

*HIV-1 matrix protein (p17)

HIV-1 Gag precursor (55 kDa) contains on its N-terminal side the matrix protein (p17 or MA) that is key viral component. Indeed, both MA myristoylation and interaction with PI(4,5)P2 are required for HIV-1 assembly at the plasma membrane [START_REF] Ono | Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane[END_REF]. Gag is thought to be cleaved by the viral protease to generate the MA and capsid (p24; CA) proteins only after budding and most Gag is unprocessed within infected cells [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF]. Nevertheless, MA unconventional secretion was observed using HIV-1 infected cells or Gag-transfected cells. This secretion was found to require PI(4,5)P2 binding. When using Gag-transfected cells, i.e. in the absence of viral protease, cellular aspartyl protease(s) cleaved the Gag precursor between MA and CA thereby allowing MA secretion [START_REF] Caccuri | Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17[END_REF] (Fig. 1). MA secretion is only observed upon expression of the complete Gag protein. Indeed, when expressed alone, MA adopts a folded conformation preventing membrane targeting [START_REF] Zhou | Differential membrane binding of the human immunodeficiency virus type 1 matrix protein[END_REF] and secretion [START_REF] Caccuri | Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17[END_REF]. Circulating MA was found to affect the function of several cell types, inducing the adhesion and migration of monocytes for instance and might therefore be involved in immune system perturbation by HIV-1 [START_REF] Giagulli | HIV-1 matrix protein p17 binds to the IL-8 receptor CXCR1 and shows IL-8-like chemokine activity on monocytes through Rho/ROCK activation[END_REF].

*Ebola virus VP40

The Ebola virus is responsible for the Ebola virus disease. The capsid protein, VP40 is a key component of the virion that is able to bind RNA, multimerize and recruit ESCRT proteins for budding [START_REF] Reynard | Unconventional secretion of Ebola virus matrix protein VP40[END_REF]. Although VP40 is devoid of signal peptide, the monomeric form of VP40 was found to be secreted in a brefeldin A-independent manner, indicating that VP40 can be unconventionally secreted. Interestingly, VP40 is recruited to the plasma membrane by PI(4,5)P2 that also enables the VP40 oligomerization required for the budding of virus-like particles [START_REF] Johnson | The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress[END_REF]. It is nevertheless not clear yet whether VP40 unconventional secretion requires PI(4,5)P2 binding. Regarding the biological significance of VP40 secretion, circulating monomeric VP40 could be responsible for the early appearance of anti-VP40 antibodies in the sera of Ebola infected patients [START_REF] Reynard | Unconventional secretion of Ebola virus matrix protein VP40[END_REF].

*Herpes virus VP22 protein

Herpes simplex virus type I is a widespread virus responsible for blisters. VP22 is a 38 kDa protein present in the tegument region, i.e. between the envelope and the capsid of the virus. VP22 does not have a signal peptide and it was found to be secreted by infected cells in a brefeldin A-independent manner [START_REF] Elliott | Intercellular trafficking and protein delivery by a herpesvirus structural protein[END_REF], thereby indicating that VP22 is secreted using an unconventional secretion pathway. Moreover, VP22 was found to be taken up by neighboring cells and concentrate into their nucleus [START_REF] Elliott | Intercellular trafficking and protein delivery by a herpesvirus structural protein[END_REF], indicating that VP22 might be used to deliver cargos intracellularly as it is the case for HIV-1 Tat [START_REF] Fawell | Tat-mediated delivery of heterologous proteins into cells[END_REF]. Indeed, VP22 was able to vectorize GFP or thymidine kinase into various cell types [START_REF] Mori | Efficient secretion of the herpes simplex virus tegument protein VP22 from living mammalian cells[END_REF]. Nevertheless, some groups were unable to reproduce these findings that seemed to be a artefact, at least in some cases [START_REF] Lundberg | Is VP22 nuclear homing an artifact?[END_REF]. Although the capacity of VP22 to transport cargos into cells is a matter of debate, VP22 secretion was confirmed in a later study showing that a fusion protein between VP22 and galactosidase was efficiently secreted [START_REF] Mori | Efficient secretion of the herpes simplex virus tegument protein VP22 from living mammalian cells[END_REF]. The mechanistic details of VP22 secretion nevertheless remains poorly documented and the possible implication of this process in viral multiplication is elusive.

*HTLV-1 Tax

Human T-cell leukemia virus type I (HTLV-I) is a retrovirus responsible for adult T-cell leukemia and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-I transactivating protein Tax (39.5 kDa) contains a leucine-rich nuclear export signal that enables its export from the nucleus to the cytoplasm, but no signal peptide. Tax concentrates into the nucleus but is also observed in the cytosol [START_REF] Bertazzoni | Intracellular localization and cellular factors interaction of HTLV-1 and HTLV-2 Tax proteins: similarities and functional differences[END_REF]. Accordingly, a Tax-YFP fusion protein essentially localized to the nucleus but was also present at the level of the ER and Golgi apparatus [START_REF] Alefantis | Secretion of the human T cell leukemia virus type I transactivator protein tax[END_REF]. Time lapse microscopy confirmed this localization of Tax-YFP in the secretory pathway. Depending on cell types 16-53% of Tax was secreted, showing that a significant fraction of cellular Tax is exported. Brefeldin A inhibited Tax export by ~60%, indicating that Tax secretion is partly but not entirely unconventional [START_REF] Alefantis | Secretion of the human T cell leukemia virus type I transactivator protein tax[END_REF]. Tax was later found, using antibody arrays, to interact with proteins regulating ER to Golgi transport ( COP and COPII), and movement from the Golgi to the plasma membrane (SNAP23, SCAMP1 and SCAMP2). Accordingly, silencing COPII, SNAP23 or SCAMP1 inhibited Tax secretion by ~60%. Sequence analysis indicated that Tax exhibits four putative secretion signals (312YTNI315, 319LL320, 330DHE332, and Val353). A point mutation approach showed that the YTNI motif is critical both for Tax localization to the exocytic pathway and for secretion [START_REF] Jain | Identification of human T cell leukemia virus type 1 tax amino Acid signals and cellular factors involved in secretion of the viral oncoprotein[END_REF]. A YXXØ motif has indeed been found to enable localization to the trans-Golgi network of transmembrane proteins that own this signal on their cytosolic tail [START_REF] Bonifacino | Angelica, Molecular bases for the recognition of tyrosine-based sorting signals[END_REF]. Nevertheless, this motif is also responsible for recognition by the AP-2 adaptor complex for clathrin-mediated receptor endocytosis [START_REF] Traub | Cargo recognition in clathrin-mediated endocytosis[END_REF] and it is therefore not clear how it could mediate the specific recruitment to the TGN of a cytosolic protein such as Tax. Tax secretion is original since it takes place despite the lack of signal sequence although largely relying on the ER/Golgi secretion apparatus (Fig. 1). Tax identified partners are present on the cytosolic leaflet of the ER, Golgi apparatus, the plasma membrane and even endosomes for COP [START_REF] Aniento | An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes[END_REF]. It is not clear yet at which stage(s) of the exocytic pathway Tax undergoes translocation to the trans side of the membrane to reach the lumen of the organelle or the outside medium (Fig. 1). The brefeldin A independent Tax secretion pathway remains to be characterized but it likely involves direct targeting to the plasma membrane, maybe following interaction with SCAMP1 or SNAP23 that concentrate at this level [START_REF] Wu | Evidence for colocalization and interaction between 37 and 39 kDa isoforms of secretory carrier membrane proteins (SCAMPs)[END_REF][START_REF] Chen | Intracellular localization of SNAP-23 to endosomal compartments[END_REF]. Tax secretion was observed using cultures of T-cells from HAM/TSP patients [START_REF] Medina | Tax secretion from peripheral blood mononuclear cells and Tax detection in plasma of patients with human T-lymphotropic virus-type 1-associated myelopathy/tropical spastic paraparesis and asymptomatic carriers[END_REF], and extracellular Tax was detected in the cerebrospinal fluid of HAM/TSP patients [START_REF] Jain | Identification of human T cell leukemia virus type 1 tax amino Acid signals and cellular factors involved in secretion of the viral oncoprotein[END_REF]. Circulating Tax indeed triggers hyperstimulation of the immune system and the release of several proinflammatory cytokines such as TNF-, IL-6 and IL-1 by microglial cells and TNF-by neurons. These cytokines might facilitate the maintenance of an inflammatory state and contribute to the demyelination of neurons that is observed during disease progression [START_REF] Jain | Identification of human T cell leukemia virus type 1 tax amino Acid signals and cellular factors involved in secretion of the viral oncoprotein[END_REF]. Secreted Tax is thus likely involved in the pathogenic processes associated with HAM/TSP.

Conclusion

Viral proteins often have multifaceted activities [START_REF] Jeang | Multifaceted activities of the HIV-1 transactivator of transcription[END_REF] and this is the case for viral proteins that are secreted using an unconventional secretion pathway. They either have a key structural role, as in the case of VP22, VP40 and MA (HIV-1) or a crucial transcriptional activity for Tax and Tat. They are additionally secreted to fulfill their extracellular or extra-viral role, which remains to be clearly established for some of them. The unconventional pathways used by viruses to achieve secretion of these proteins are diverse (Fig. 1). They nevertheless all seem to be self-translocating proteins, indicating that they follow a type I pathway of unconventional secretion [START_REF] Rabouille | Pathways of Unconventional Protein Secretion[END_REF]. This is particularly clear for HIV-Tat which secretion process showed several similarities with that of FGF2, another well studied Type I UPS cargo [START_REF] Steringer | Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components[END_REF]. There is a striking common membrane targeting molecule, PI(4,5)P2 that is required for the secretion of Tat [START_REF] Rayne | HIV-1 Tat is unconventionally secreted through the plasma membrane[END_REF] FGF2 [START_REF] Steringer | Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion[END_REF], MA [START_REF] Caccuri | Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17[END_REF], and for VP40 membrane association [START_REF] Johnson | The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress[END_REF]. It is still not known whether Tat, MA and VP40 secretion exhibit other similarities. A common characteristic of all these viral proteins secreted using unconventional pathways is that they are all expressed early upon cell infection [START_REF] Elliott | Intercellular trafficking and protein delivery by a herpesvirus structural protein[END_REF][START_REF] Das | The HIV-1 Tat protein has a versatile role in activating viral transcription[END_REF][START_REF] Giam | HTLV-1 Tax and adult T-cell leukemia[END_REF][START_REF] Radzimanowski | Conformational plasticity of the Ebola virus matrix protein[END_REF], indicating that secretion can potentially begin as soon as viral protein synthesis starts. Overall these secretion processes are poorly documented. For instance, whether cellular proteins facilitate or regulate these unconventional secretion pathways is still unclear. Further work should enable to understand how these viral proteins can cross membranes to reach the extracellular milieu.

Figure 1.

Unconventional secretion pathways of viral proteins. HTVL-I Tax can be secreted both by a brefeldin A dependent route, i.e. following the conventional ER/Golgi pathway or by a brefeldin Ainsensitive pathway, presumably involving direct transmembrane transport. HIV-1 Tat is exported directly through the plasma membrane following PI(4,5) P2-mediated recruitment at this level (inset). HIV-1 matrix protein (MA; p17) secretion requires first binding of the Gag precursor to PI(4,5)P2, then proteolysis by the viral or a cellular protease to release p17 from p24 (the capsid protein), enabling p17 export. VP40 is also recruited at the plasma membrane by PI(4,5)P2, and was found to be unconventionally secreted. VP22 secretion mechanism is poorly documented. See the text for details. TGN, trans Golgi network.

Acknowledgements

We are indebted to JM Mesnard and JM Peloponese (IRIM, Montpellier) for advice on HTLV-1, and O. Reynard (CIRI, Lyon) for discussions on VP40. Our group is funded by the CNRS and the FRM (Equipe FRM 20161136701).