NEW INSIGHTS INTO HUMAN IMMUNODEFICIENCY VIRUS - TYPE 1 REPLICATION B. Grigorov1,2, D. Muriaux1, R. Argirova2, Jean-Luc Darlix1 LaboRétro-Unité de Virologie Humaine, U412 Inserm, ENS de Lyon, Lyon, France1 National Center of Infectious and Parasitic Diseases, Laboratory of Retroviruses, Sofia, Bulgaria2

ABSTRACT Retroviruses comprise a large and widespread family of enveloped plus strand RNA viruses, which can infect most if not all vertebrates and cause diverse pathologies such as leukemia, sarcoma, neurodegeneration and immunodeficiency. The human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and a member of the Retroviridae family. Investigating the different steps of HIV-1 replication and the interactions of HIV-1 with components of the infected cell is of critical importance for the identification of novel therapeutic approaches and agents. In this review, we describe the early events of HIV-1 replication, namely how the virus recognizes and enters the target cell, the replication strategy and interactions between viral and cellular components and, the late stages corresponding to assembly and budding of progeny virions which necessitate viral proteins, RNA and cellular membranes. In addition, we report on the recent discoveries of cellular innate defense mechanisms against HIV-1.

Introduction

virus assembly and structure. Along this line, this mini-review summarizes recent data on HIV-1 entry and genome replication and provides new insights into HIV-1 assembly and budding.

HIV-1 is the causative agent of AIDS (acquired immune deficiency syndrome), classified as a lentivirus member of the Retroviridae family. According to WHO more than 42 millions people are infected by HIV-1 worldwide in 2004. This is why the fight against AIDS using multi-therapies (HAART) is such an important issue. HAART is based on reverse transcriptase (RT) and protease (PR) inhibitors and is effective but can be rather detrimental to HIV infected persons due to side effects. In addition, emergence and dissemination of HIV-1 strains resistant to RT and PR inhibitors is a growing concern. Therefore, the search for new RT and PR inhibitors and novel drugs acting on structural elements of HIV requires, needless to say, a better understanding of the structure of the virus basic components that are Gag, Env and the genomic RNA, of their functions in genome replication, and implications in

HIV-1 genetic structure The HIV-1 genome is present as two positive-stranded RNA molecules tightly associated in the form of a 60S complex within the viral particle. The two genome RNAs can be identical, thus corresponding to a homozygous virus, or different corresponding to a heterozygous virus. The genomic RNA is converted into a double stranded proviral DNA flanked by two long terminal repeats (LTR) by the viral DNA polymerase RT assisted by the viral nucleocapsid protein NC. During the conversion of RNA to DNA by RT, point mutations can be incorporated at a high rate due to the lack of RT proof reading activity. In addition, gene rearrangements by recombi3

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Fig. 1A. HIV DNA genetic structure.

Fig. 1B. HIV particle morphology.

nation can occur when RT copies the genome of heterozygous virions. Both point mutations and recombination reactions contribute to the high genetic variability of HIV-1 and the rapid emergence of strains resistant to HAART (4). The Biotechnol. & Biotechnol. Eq. 19/2005/1

proviral DNA is subsequently integrated into active regions of the cellular genome by the viral enzyme Integrase (IN) assisted by NC. The proviral DNA (Fig. 1) with the LTR contains three major retroviral genes which 4

Fig. 2.

are gag (coding for the viral structural proteins matrix MAp17, capsid CAp24, nucleocapsid NCp7, and p6), pol (coding for the viral enzymes protease (PR), reverse transcriptase (RT) and integrase (IN), and env (encoding the surface SUgp120 and transmembrane TMgp41 glycoproteins) (Fig. 1A). HIV-1 also encodes nonstructural key regulatory factors which are Vif (the virus infectivity factor; see below), Nef (downregulating CD4 expression and MHC I presentation and enhancing infectivity), Rev (regulating genomic RNA splicing by promoting nuclear export of the genomic and Env RNA), Tat (the transactivator of HIV-1 provirus transcription), Vpr (causing a G2/M arrest of infected cells) and Vpu (causing CD4 degradation and improving viral budding). In addition, the viral genome contains series of cis-acting sequences (Fig.1.), such as the Tat trans-activating region (TAR), the internal ribosome entry site (IRES), the primer tRNA binding site (PBS), the dimer initiation site (DIS), the packaging sequence (Psi), the polypurine tract (PPT located at the 3’ end of the genome and a

second cPPT present in the middle of the genome) and the Rev-responsive element (RRE). These signals play critical roles in the viral life cycle such as Provirus transcription (TAR), nuclear export (RRE) of the genomic RNA, its translation (IRES), dimerization (DIS) and packaging (Psi) into a newly formed particle, and genomic RNA to proviral DNA conversion (PBS, PPT and cPPT)(26,28).

HIV-1 Replication Entry, viral DNA synthesis and the dual roles of cellular proteins HIV-1 infection starts when virions either on their own or presented by infected cells bind a target cell such as macrophage or CD4+ T lymphocyte (Fig. 2). Entry of HIV first requires recognition of the cellular CD4 receptor by SUgp120 molecules and binding and, next, a second recognition and binding event involving a co-receptor, CCR5 or CXCR4, in most cases (7). Among many classifications, HIV-1 has been categorized in T-tropic, M-tropic an dual M/T tropic strains according to the cells they infect and the co-receptor used to 5

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that end. T tropic viruses infect CD4+ Tcells and use the chemokine CXCR4 as coreceptor and are syncytium inducing (SI). M-tropic viruses infect macrophages, utilize the CCR5 co-receptor (1) and are nonsyncytium inducing (NSI). M/T tropic HIV strains can infect both types of cells. HIV-1 infects Langerhans cells (LC) and dendritic cells (DC) both in vivo and in vitro (101), although inefficiently compared with HIV-1 infection of CD4+T cells. Allowing a low level of viral replication, DCs can capture HIV-1 particles, without necessarily becoming infected, and transfer the infectious virus to CD4+ T cells which become productively infected (11,112). In immature DC, the C-type lectin DC-SIGN (also known as CD209) is highly expressed and is a major actor mediating HIV-1 attachment to DCs and transfer to T cells (36). Other subsets of DCs, such as LCs, do not express detectable levels of DC-SIGN but are nevertheless able to capture and transfer HIV-1 infection via binding to other lectins such as the mannose receptor and langerin (48,73). Adhesion molecules on DCs can recognize cognate ligands (such as ICAM-1), which are incorporated into the envelope of HIV1 virions during the budding process(109). DC-SIGN has been also shown to function as an attachment factor for HIV-2, simian immunodeficiency virus (6,88), cytomegalovirus (44), Dengue virus (110), Ebola virus (3), and SARS-Corona virus (119). Interestingly, some primary M/T tropic HIV-1 isolates, especially those obtained from early sero-converters, can efficiently infect primary astrocytes, endothelial cells and macrophages through the use of alternative co-receptors such as D6, a promiscuous chemokine receptor highly expressed on these cell types (Neil et al. unpublished data). The affinity of HIV-1 SUgp120 glycoprotein for the cellular CCR5 protein is greatly enhanced in the presence of CD4, emphasizing that CD4 not only provide a Biotechnol. & Biotechnol. Eq. 19/2005/1

docking surface for HIV-1 through SUgp120 but also promotes exposure of a SU domain that interacts with chemokine receptors (111,118). Chemokine-receptor binding triggers conformational changes in SUgp120 leading to the exposure of the fusogenic peptide at the amino-terminus of the TMgp41 glycoprotein (55). This, in turn, modifies TMgp41 into its fusion-active state whereby it inserts into the target membrane (55,65). Thus, the viral envelope and plasma membrane fuse via a direct, pH-independent mechanism, thereby releasing the viral core into the target cell cytoplasm to initiate replication (102). After entry of the viral core into the cytoplasm, the genomic RNA is converted into a double stranded proviral DNA by RT chaperoned by NC at the levels of initiation and of the two obligatory strand transfers to generate the LTR (28). Recently, a mechanism by which cells can restrict HIV-1 infection was discovered. Indeed Sheehy et al. (2002)(98) reported that HIV-1 Vif protein interacts with the cellular enzyme cytidine deaminase APOBEC3G (Fig. 3A). In the absence of Vif, APOBEC3G is incorporated into HIV1 virions and acts during reverse transcription to convert cytosine residues to uracils in the newly made cDNA. This results in the degradation of the neo-synthesized cDNA through the action of host glycosidases and repair enzymes. This can also lead to G-to-A hypermutations in the newly made viral DNA. But, Vif interfers with the antiviral activity of APOBEC3G by blocking its incorporation into virions produced by infected cells. Since the virion incorporation of APOBEC3G is required for its antiviral activity, several groups have focused on how this cellular protein can get into virions. It was suggested that APOBEC3G directly interacts with Gag via its NC domain (2), or else with the genomic RNA since it is one of the two viral assembly platforms (see below). Based on these recent findings, one can 6

Fig. 3.

speculate that APOBEC3G has evolved as a defense mechanism against HIV/SIV infection and that Vif can be viewed as a new viral target for antiviral drug development (71). It has long been known that retroviral infection can be restricted by cellular factors such as Fv-1-mediated restriction of murine leukemia virus replication in some mouse cells, acting on the capsid of incoming MLV particles (10,106). More recently, HIV-1 was found to poorly replicate in non-human primate cells (72) and viceversa SIV poorly infects human cells. This led to the identification of lentiviral (LV) restriction factors, which appear to act at the level of incoming capsid particles (45), such a restriction factor has recently been discovered and found to be TRIM-5α. In agreement with the original observations,

HIV-1 infection is more contained by rhesus monkey TRIM5alpha than by the human homolog. Thus TRIM 5α can be considered as a protein, which together with other cellular factors mediate anti-viral innate defenses in order to restrict virus entry and replication (105). The TRIM (Tripartite Motif) protein family consists of more than 37 members (94). TRIM proteins are concentrated in “cytoplasmic” bodies of variable size, occasionally located around the nucleus. TRIM is composed of three Zn binding domains: a RING (R), a B-box type 1 (B1) and a Bbox type 2 (B2), followed by a coiled coil (CC) region (15,92) (Fig.3A.). Genes belonging to this family are implicated in a variety of processes, such as cell growth and development, and are implicated in several human diseases. Innate anti-lentivi7

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ral defense would add another function to these proteins. Integration of HIV-1 proviral DNA and expression Following reverse transcription, the newly synthesized DNA flanked by two LTR is contained within a poorly defined preintegration complex (PIC) that is imported into the nucleus. PIC components are thought to involve viral proteins such as RT, IN, NC, MA and Vpr (18,32,35) and cellular factors stimulating proviral integration such as BAF (barrier to auto-integration factor) (57,107), the high motility group proteinHMGa1 (Farnet et al. 1997), the IN-interactor 1 (Ini-1) protein which is a component of the SNF–SWI (sucrose non-fermenting–switch) complex involved in chromatin remodelling (52) and the Lens epithelium-derived growth factor (LEDGF/p75) important for nuclear localization of IN (24). PIC import into the nucleus is independent on nuclear envelope breakdown since virus replication can occur in nondividing cells (37,116). In fact, HIV-1 productively infects terminally differentiated non-dividing cells such as macrophages and dendritic cells, a property important for viral dissemination and transmission in vivo (18,19,66). Viral factors involved in HIV-1 PIC entry into the nucleus include Vpr and MA, which independently permit the import of the viral PIC through the nuclear pore via distinct nuclear localization signals (35,46,115,120). Integrase (IN) is the retroviral enzyme responsible for integration of the proviral DNA into a cell chromosome. It has been suggested that HIV-1 IN, as an essential component of the PIC, plays a role in its nuclear import (30,87). The mechanism of HIV-1 IN nuclear import, however, has not yet been fully elucidated. Recently, it has been found that LEDGF/p75 protein forms a specific nuclear complex with HIV-1 IN and is essential for nuclear localization and chromosomal association of the viral proBiotechnol. & Biotechnol. Eq. 19/2005/1

tein (62). Once inside the nucleus, the viral DNA is integrated into the host chromosome at non-specific loci by IN(18,19). More recently, it has been shown that active genes are favored integration sites for HIV-1 in primary cells and transformed cell lines (67). At this stage the proviral DNA is named provirus. The HIV-1 provirus is transcribed by the cellular host machinery to generate the genome length RNA which originates from a single promoter in the U3 region of the 5’ LTR. The first viral RNAs to be present in the cytoplasm are multiply spliced mRNAs, encoding the regulatory proteins Tat, Rev and Nef. Upon nuclear import, Tat transactivates provirus transcription by host RNA-polymerase-II via Tat-TAR interactions (113). Rev also gets into the nucleus from which it directs export of the genome length RNA and Env mRNA via Rev-RRE interactions (63). Nef is synthesized in large quantities and is found on the cytosolic membrane causing the down-regulation of antigen presentation by MHC class I. In addition Nef causes internalization of the CD4 receptor. Both Nef-directed mechanisms contribute to protect, at least in part, infected cells from the immune system ( 86,100). HIV protein R (Vpr) also accumulates in infected cells which, at the same time, promotes a G2/M arrest of these cells and activates provirus transcription. Translation of the genome length RNA to synthesize Gag and Gag-Pol polyproteins appears to take place by an IRES-dependent mechanism. Unlike the canonical 5’ Cap-dependent ribosome scanning mechanism, the IRES mechanism postulates that cellular ribosomes have a direct access to the initiator codon of a coding sequence (17). In the case of HIV, ribosomes have a direct internal entry to the Gag AUG start codon by means of structures present in the 5’ untranslated leader and at the 5’ end of Gag. This IRES-dependent mechanism first discovered in Picornaviruses also applies to other Lentiviruses (75) and Oncoretrovi8

ruses MLV, SNV and ASLV (9). The IRES mechanism is most effective in the G2/M phase of the cell cycle since during that period 5‘Cap translation is severely inhibited (91). Consequently, both Vpr blocking infected cells in G2/M and the HIV-1 IRES appear to ensure a high level of Gag and Gag-Pol synthesis.

and folding into stem-loop structures (60, 61). The NC domain of Gag is composed of two zinc fingers flanked by basic domains and the zinc fingers were found to direct genomic RNA selection and packaging (8, 27, 28, 60, 61, 69, 89). More generally RNA appears to be an essential determinant of virus assembly since complete deletion of Gag NC caused a drastic reduction in virus production (21,28,70). This favors the notion that assembly is initiated by Gag and Gag-Pol oligomerization upon their binding to the genomic RNA via NC. Next, it is assumed that binding of Gag molecules to an RNA causes a conformational switch of the Nmyristate of Gag, from hidden to exposed and thus driving the anchoring of Gag into a cellular membrane (108). The very C-terminal domain of HIV-1 Gag is p6 which contains proline-rich motifs and is required for efficient virus release (41,49,117). These motifs have been named late or L domains because virus assembly is arrested at the late stages when they are mutated (33). Yet, three different classes of late domains have been characterized (Table) which bind different cellular factors (12,16,42,64,104). Recent findings suggest that retroviral Gag proteins recruit distinct subsets of the cellular multiprotein complex ESCRT (endosomal-associated complex required for transport) necessary for endosomal protein sorting, and use it for virus budding and release (see section below). Where and how is assembly taking place? It is unclear where Gag, Gag-Pol, Env and the genomic RNA meet and interact to produce infectious viral particles. Originally electronic microscopic studies of infected lymphocytes or T-cell lines have indicated that lentiviruses can assemble and bud at the plasma membrane (5, 38). On the other hand, intracellular HIV was observed in cytoplasmic compartments, notably in macrophages (77) (Fig. 4). Recently, several studies described HIV-1 budding

HIV-1 Assembly and Budding To ensure efficient virus assembly and release, Gag, Gag-Pol, the genomic RNA and the envelope glycoproteins should meet in a coordinated manner. As reviewed below, the emerging picture of these coordinated macromolecular interactions proposes that Gag orchestrates assembly and to that end utilizes two platforms that are the genomic RNA as a hydrophilic platform and a cellular membrane as a lipid platform. It is also proposed that a major part of virus assembly is taking place in intracellular vesicles and that viral particles exploit the vesicular sorting pathway to leave the infected cell. The major viral actors Gag is the sole structural protein in Retroviruses and as such orchestrates viral particle assembly and release. However, in the absence of the genomic RNA of the enzymes and the glycoproteins components, only non infectious virus-like particles (VLPs) are formed (39,95). Thus, Gag contains all the informations necessary to govern the assembly and release processes and is considered to be the major actor in retrovirus assembly. Gag also directs the dimerization and specific packaging of the RNA genome and the recruitment of viral proteins, such as Vpr and Vif, as well as host-cell factors into newly formed virus particles (reviewed in 22). Selective and efficient genomic RNA packaging is driven by molecular interactions between the NC domain of Gag and specific cis-acting RNA sequences termed ‘packaging signal’ or Psi, located in the untranslated 5’ leader of the genomic RNA 9

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TABLE Retroviral late domains, and cellular factors with which they interact Late domain Retrovirus

Recruited cellular factors

References

HIV-1 HTLV-1 (human T-lymphotropic virus), MPMV (Mason-Pfizer monkey virus)

TSG101-sorting into MVB’s

41, 49 29

HIV-1, EIAV (equine infectious anemia virus)

AIP1/Alix-MVB vesicles formation

104

YP(x)nL

RSV

Nedd4 family of ubiquitin E3 ligases

PPxY

(Rous sarcoma virus), MoMLV (Moloney murine leukemia virus), HTLV-1,

P(T/A)AP

MPMV

80, 122 12, 16 121

Fig. 4. Electron microscopy thin section of 293J cells expressing HIV-1.

and accumulation in late endosomes, suggesting that HIV assembly and budding are taking place intracellularly as well as at the plasma membrane. Therefore the present view is that cellular membranes act as an indispensable platform for HIV assembly. More specifically, Gag molecules anchored into membranes were found to accumulate in specific microdomains, called lipid rafts that are small detergent-resistant membrane fractions. They are composed of clusters of sphingolipids and cholesterol that can Biotechnol. & Biotechnol. Eq. 19/2005/1

move within the fluid bilayer (reviewed in 99). HIV-1 was proposed to utilize lipid rafts as membrane platforms for assembly and virion release as indicated by series of data (23): (i) raft-associated molecules, including sphingomyelin, phosphatidylethanolamine and cholesterol are enriched in HIV particles (74,76). This may reflect, at least in part, the fact that retroviruses can bud into cholesterol-rich MVB membranes (68); (ii) several HIV-1 proteins such as Gag, Gag-Pol and Vif are enriched in de10

tergent-resistant membrane fractions characteristic of rafts (47,59) and (iii) cholesterol depletion inhibits virus release (20,58). Interestingly, the cellular prion protein (PrPc) which is anchored into lipid rafts was found to interact with HIV GagNC in vitro (34) and to interfere with HIV1 assembly (56). Indeed, PrPc overexpression caused a more than tenfold decrease of virus production and viruses were less infectious. Mutating PrPc to prevent its association with membranes relieved its negative impact on HIV-1 assembly and infectivity. These findings clearly suggest a role of PrPc in a membrane-dependent innate immunity against HIV-1, the mechanism of which remains to be elucidated. Intracellular, membrane-directed assembly of HIV is most probably useful for envelope glycoproteins (Env) recruitment by Gag. In fact, the cytoplasmic domain of TMgp41 contains a highly conserved tyrosine-based motif that can mediate endocytosis of Env through clathrin-coated pits (31,96). The presence of this signal results in the majority of Env being located in intracellular membranes. Gag itself may also undergo endocytosis as a trafficking step between the plasma membrane and MVB as it was recently shown that HIV-1 Gag contains a dileucine-like motif that regulates its targeting to multi-vesicular body (MVB) (59). The possibility that Gag and Env meet on intracellular membranes, which may differ according to the cell type is still an open question. In monocyte-derived macrophages, HIV assembles mainly in intracellular vesicles containing markers for late endosomes/MVBs. As the virus buds into this compartment, it acquires various cellular proteins characteristic of late endosomes/MVBs, including the CD63 and CD81 tetraspanins and some LAMP-1 (Lysosome-associated membrane proteins) (83). The budding of HIV uses host cell machineries that normally function in the formation of the MVB and small intracellular vesicles. HIV particles budding at the

cell surface would need to redirect these so-called ESCRT complexes to the plasma membrane, whereas the presence of the ESCRT complexes on endosomes may favor intracellular assembly. Along this line, ubiquitin is an important sorting factor for endocytosis and early endosomal protein trafficking (43). Ubiquitination is necessary and sufficient to direct many proteins from the endosomal membrane into MVBs (53). As a result many proteins that enter the MVB pathway may already be ubiquitinylated, such as retroviral Gag proteins (78). Thus, ubiquitin transfer might play a role in the release of enveloped viruses that utilize PTAP and PPXY late domains (33). This is supported by the fact that HIV-1 and MuLV Gag proteins are mono-ubiquitylated at multiple sites (78). Moreover, some mutations in L domain prevent Gag ubiquitination and can also inhibit virus budding (103). Altogether, these observations indicate that HIV-1 Gag ubiquitiation is probably a sorting signal conducting Gag through the MVB pathway. Thus, intracellular assembly of HIV would hide the virus and provide protection against the immune system. Virus maturation and Core condensation Concomitant with Gag and Gag-Pol assembly, recruitment of the envelope glycoproteins and formation of new viral particles, processing of Gag and Gag-Pol precursors by the viral PR takes place, which triggers major changes in virion composition and morphology (Fig.1B). These processes are known as virus maturation and core condensation, during which Gag and Gag-Pol proteins are processed into the mature structural proteins and enzymes and the genomic RNA undergoes condensation in the form of a 60S complex by mature NC protein molecules (28,93). Maturation is not required per se for particle assembly but it accelerates virion production and is essential for virus infectivity (54,84). Mature infectious virions get released from 11

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infected cells, either by fusion of viruscontaining endosomes with the plasma membrane or directly by particle budding from the plasma membrane into the extracellular medium for new rounds of infection. It is noteworthy that presence of an active viral PR in HIV infected cells can have dramatic consequences on cell survival since PR was found to cleave the major factor of translation initiation, namely eIF4G, which subsequently inhibits Cap-dependant translation and promotes cell death (90). This may explain, at least in part, why antiPR inhibitors used in HAART provide such a benefit in AIDS treatments.

and T cell-T cell (51). The virological synapse could be accommodated within any comprehensive model for retrovirus release (68). The transfer of HIV-1 at the T cell synapse requires viral budding (rather than cell-cell fusion) (51) and therefore presumably employs the same set of factors facilitating virus budding through intracellular and plasma membranes. Interestingly, intact vrions that are taken up into immature dendritic cells by the DC-SIGN receptor, concentrate at intracellular sites that are positive for MVB markers (T. Hope, personal communication). It seems likely that in order to be released into the synapse, these particles usurp the existing cellular pathways that are normally used to transport late endosomal/MVB compartments to sites of T cell contact upon DC maturation (13,25).

The Virological Synapse and Virus Dissemination in vivo Interestingly, retroviruses can be preferentially released at sites of cell-to-cell contact, a discovery that has major implications for the efficiency of viral spread in vivo. Both HIV-1 and HTLV-1 particles have been shown to transfer directionally through sites of contact between infected and uninfected T cells (50,51). Cell-to-cell transmission favors HIV-1 replication because it obviates the rate-limiting step of virus diffusion prior to attachment and might reduce or prevent virus neutralization by antibodies and complement in vivo. The sites of cell-to-cell contact that mediate virus transfer have been termed virological synapse (50,51). The virological synapse (VS) meets the criteria for justifying the use of “synapse” because cells contact each other but remain individual entities (unfused), and a stable adhesive junction forms between them. In addition, the export of viral material from the pre- to the postsynaptic side of this junction is directed across the synapse by cellular machinery, which in the case of the VS involves the transfer of viral particles from the effector infected cell, to the target cell expressing the appropriate receptors. In the case of HIV-1 infection, two types of VS have been described: dendritic cell-T cell (112) Biotechnol. & Biotechnol. Eq. 19/2005/1

Conclusions During the two decades following HIV-1 discovery, research was mainly focused on the characterization of the major viral structural and enzymatic components involved in virus replication and on specific immune responses directed at these components. Nowadays, HIV-1 research has fully taken into account the fact that retroviruses like HIV act as cell parasites which need to interact with and divert cellular machineries and organelles to replicate and disseminate. This will open the way to better characterizing and understanding, with sometimes a flavor of revisiting ancient notions and findings, HIV assembly and dissemination by means of cell-to-cell contacts, and with the hope of finding new antiviral drugs.

Acknowledgements We would like to thank INSERM, ANRS, European TRIoH Consortium, the French Embassy in Bulgaria and The National Institute of Infectious and Parasitic DiseasesSofia for their support. 12

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