HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells.

BACKGROUND: Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized. RESULTS: Here, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs. CONCLUSION: Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.

The infectious synapse formed between mature dendritic cells and CD4(+) T cells is independent of the presence of the HIV-1 envelope glycoprotein.

BACKGROUND: Since cell-mediated infection of human immunodeficiency virus type 1 (HIV-1) is more efficient than cell-free infection, cell-to-cell propagation plays a crucial role in the pathogenesis of HIV-1 infection. Transmission of HIV-1 is enabled by two types of cellular contacts, namely, virological synapses between productively infected cells and uninfected target cells and infectious synapses between uninfected dendritic cells (DC) harboring HIV-1 and uninfected target cells. While virological synapses are driven by expression of the viral envelope glycoprotein on the cell surface, little is known about the role of envelope glycoprotein during contact between DC and T cells. We explored the contribution of HIV-1 envelope glycoprotein, adhesion molecules, and antigen recognition in the formation of conjugates comprising mature DC (mDC) and CD4(+) T cells in order to further evaluate their role in mDC-mediated HIV-1 transmission at the immunological synapse. RESULTS: Unlike virological synapse, HIV-1 did not modulate the formation of cell conjugates comprising mDC harboring HIV-1 and non-activated primary CD4(+) T cells. Disruption of interactions between ICAM-1 and LFA-1, however, resulted in a 60% decrease in mDC-CD4(+) T-cell conjugate formation and, consequently, in a significant reduction of mDC-mediated HIV-1 transmission to non-activated primary CD4(+) T cells (p < 0.05). Antigen recognition or sustained MHC-TcR interaction did not enhance conjugate formation, but significantly boosted productive mDC-mediated transmission of HIV-1 (p < 0.05) by increasing T-cell activation and proliferation. CONCLUSIONS: Formation of the infectious synapse is independent of the presence of the HIV-1 envelope glycoprotein, although it does require an interaction between ICAM-1 and LFA-1. This interaction is the main driving force behind the formation of mDC-CD4(+) T-cell conjugates and enables transmission of HIV-1 to CD4(+) T cells. Moreover, antigen recognition boosts HIV-1 replication without affecting the frequency of cellular conjugates. Our results suggest a determinant role for immune activation driven by mDC-CD4(+) T-cell contacts in viral dissemination and that this activation likely contributes to the pathogenesis of HIV-1 infection.

SAMHD1 restricts HIV-1 cell-to-cell transmission and limits immune detection in monocyte-derived dendritic cells.

SAMHD1 is a viral restriction factor expressed in dendritic cells and other cells, inhibiting infection by cell-free human immunodeficiency virus type 1 (HIV-1) particles. SAMHD1 depletes the intracellular pool of deoxynucleoside triphosphates, thus impairing HIV-1 reverse transcription and productive infection in noncycling cells. The Vpx protein from HIV-2 or simian immunodeficiency virus (SIVsm/SIVmac) antagonizes the effect of SAMHD1 by triggering its degradation. A large part of HIV-1 spread occurs through direct contacts between infected cells and bystander target cells. Here, we asked whether SAMHD1 impairs direct HIV-1 transmission from infected T lymphocytes to monocyte-derived dendritic cells (MDDCs). HIV-1-infected lymphocytes were cocultivated with MDDCs that have been pretreated or not with Vpx or with small interfering RNA against SAMHD1. We show that in the cocultures, SAMHD1 significantly inhibits productive cell-to-cell transmission to target MDDCs and prevents the type I interferon response and expression of the interferon-stimulated gene MxA. Therefore, SAMHD1, by controlling the sensitivity of MDDCs to HIV-1 infection during intercellular contacts, impacts their ability to sense the virus and to trigger an innate immune response.

Viral infection: Moving through complex and dynamic cell-membrane structures.

Viruses have developed different survival strategies in host cells by crossing cell-membrane compartments, during different steps of their viral life cycle. In fact, the non-regenerative viral membrane of enveloped viruses needs to encounter the dynamic cell-host membrane, during early steps of the infection process, in which both membranes fuse, either at cell-surface or in an endocytic compartment, to promote viral entry and infection. Once inside the cell, many viruses accomplish their replication process through exploiting or modulating membrane traffic, and generating specialized compartments to assure viral replication, viral budding and spreading, which also serve to evade the immune responses against the pathogen. In this review, we have attempted to present some data that highlight the importance of membrane dynamics during viral entry and replicative processes, in order to understand how viruses use and move through different complex and dynamic cell-membrane structures and how they use them to persist.

HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes.

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6-guanosine 5'-diphosphate/guanosine 5'-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope-induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4(+) T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate-associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G-pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4(+) T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

Could CD4 capture by CD8+ T cells play a role in HIV spreading?

CD8(+) T cells have been shown to capture plasma membrane fragments from target cells expressing their cognate antigen, a process termed "trogocytosis". Here, we report that human CD4, the Human Immunodeficiency Virus (HIV) receptor, can be found among the proteins transferred by trogocytosis. CD4 is expressed in a correct orientation after its capture by CD8(+) T cells as shown by its detection using conformational antibodies and its ability to allow HIV binding on recipient CD8(+) T cells. Although we could not find direct evidence for infection of CD8(+) T cells having captured CD4 by HIV, CD4 was virologically functional on these cells as it conferred on them the ability to undergo syncytia formation induced by HIV-infected MOLT-4 cells. Our results show that acquisition of CD4 by CD8(+) T cells via trogocytosis could play a previously unappreciated role for CD8(+) T cells in HIV spreading possibly without leading to their infection.

On the steps of cell-to-cell HIV transmission between CD4 T cells.

Although cell-to-cell HIV transmission was defined in early 90's, in the last five years, several groups have underscored the relevance of this mode of HIV spread between productively infected and uninfected CD4 T cells by defining the term virological synapse (VS). However, unraveling the molecular mechanisms of this efficient mode of viral spread appears to be more controversial than expected. Different authors have highlighted the role of a classical co-receptor-dependent HIV transmission while others describe a co-receptor-independent mechanism as predominant in VS. By analyzing different cellular models (primary cells and cell lines), we suggest that primary cells are highly sensitive to the physical passage of viral particles across the synapses, a co-receptor-independent phenomenon that we call "HIV transfer". Once viral particles are transferred, they can infect target cells by a co-receptor-dependent mechanism that fits with the classical meaning of "HIV transmission" and that is much more efficient in cell lines. Differences in the ability of primary CD4 T cells and cell lines to support HIV transfer and transmission explain most of the reported controversial data and should be taken into account when analyzing cell-to-cell HIV spread. Moreover, the terms transfer and transmission may be useful to define the events occurring at the VS. Thus, HIV particles would be transferred across synapses, while HIV infection would be transmitted between cells. Chronologically, HIV transfer is an early event occurring immediately after the VS formation, which precedes but does not inevitably lead to transmission, a late event resulting in infection.

Antigp41 antibodies fail to block early events of virological synapses but inhibit HIV spread between T cells.

OBJECTIVE: Compared with cell-free viral infection, virological synapses increase HIV capture by target cells, viral infectivity and cytopathicity, and are believed to be less sensitive to antibody neutralization. We have evaluated the impact of antibodies against HIV envelope glycoproteins (gp120 and gp41) on cell-to-cell HIV transmission. METHODS: We analyzed the role of trogocytosis in cell-to-cell HIV transmission and the inhibitory mechanisms of antigp120 antibody IgGb12 and antigp41 antibodies 4E10 and 2F5 using cocultures of NL4-3 or BaL-infected MOLT/CCR5 cells with primary CD4 T cells. RESULTS: Analysis of early steps of HIV transmission in these cocultures showed that IgGb12, but not 4E10 and 2F5, inhibited the formation of virological synapses. Consequently, IgGb12 but not antigp41 antibodies blocked the transfer of HIV particles from infected to target cells and the trogocytic transfer of CD4 molecules from target to infected cells. Interestingly, trogocytic transfer of membranes was not detected in the HIV transmission direction. Furthermore, analysis of late events of HIV transmission showed that all neutralizing antibodies blocked productive infection of target cells, suggesting that HIV infection between T cells is transmitted by a neutralization-sensitive mechanism involving HIV budding from infected cells and capture by target cells. CONCLUSION: Despite mechanistic differences, antigp120 and antigp41 antibodies block infectious cell-to-cell HIV transmission. Our data suggest that eliciting high titers of neutralizing antibodies in vivo should be maintained as a main end of HIV vaccine design.

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4.

BACKGROUND: Cell-to-cell HIV transmission requires cellular contacts that may be in part mediated by the integrin leukocyte function antigen (LFA)-1 and its ligands intercellular adhesion molecule (ICAM)-1, -2 and -3. The role of these molecules in free virus infection of CD4 T cells or in transinfection mediated by dendritic cells (DC) has been previously described. Here, we evaluate their role in viral transmission between different HIV producing cells and primary CD4 T cells. RESULTS: The formation of cellular conjugates and subsequent HIV transmission between productively infected MOLT cell lines and primary CD4 T cells was not inhibited by a panel of blocking antibodies against ICAM-1, ICAM-3 and alpha and beta chains of LFA-1. Complete abrogation of HIV transmission and formation of cellular conjugates was only observed when gp120/CD4 interactions were blocked. The dispensable role of LFA-1 in HIV transmission was confirmed using non-lymphoid 293T cells, lacking the expression of adhesion molecules, as HIV producing cells. Moreover, HIV transmission between infected and uninfected primary CD4 T cells was abrogated by inhibitors of gp120 binding to CD4 but was not inhibited by blocking LFA-1 binding to ICAM-1 or ICAM-3. Rather, LFA-1 and ICAM-3 mAbs enhanced HIV transfer. All HIV producing cells (including 293T cells) transferred HIV particles more efficiently to memory than to naive CD4 T cells. CONCLUSION: In contrast to other mechanisms of viral spread, HIV transmission between infected and uninfected T cells efficiently occurs in the absence of adhesion molecules. Thus, gp120/CD4 interactions are the main driving force of the formation of cellular contacts between infected and uninfected CD4 T cells whereby HIV transmission occurs.

Association between HIV replication and cholesterol in peripheral blood mononuclear cells in HIV-infected patients interrupting HAART.

BACKGROUND: Cellular cholesterol is essential for HIV replication and may control HIV spread. HIV, in turn, appears to control cholesterol metabolism. OBJECTIVES: To describe the relationships between serum lipids, cellular cholesterol and viral replication during highly active antiretroviral therapy (HAART) interruption. METHODS: We have correlated virological parameters with the level of circulating lipids in serum and the content of cellular cholesterol in peripheral blood mononuclear cells (PBMCs). The study included 33 patients interrupting HAART with (n = 23) or without (n = 10) atorvastatin treatment. RESULTS: Atorvastatin treatment did not modify PBMC cholesterol levels at week 4 after HAART interruption, although it significantly reduced serum cholesterol (total and LDL, where LDL stands for low density lipoprotein) (P < 0.05). Serum cholesterol or LDL marginally influenced PBMC cholesterol since no significant correlations were found between these parameters either at 0 or 4 weeks after HAART interruption. Analysis of virological data in all patients revealed a negative trend (P = 0.07) between baseline PBMC cholesterol and absolute CD4 T cell counts at baseline but a poor correlation (P = 0.18) with the viral load (VL) at week 4. Separate analysis of control patients showed a correlation between baseline PBMC cholesterol and VL at week 4 (P = 0.01). However, atorvastatin treatment abrogated this correlation by increasing viral replication in individuals with low cellular cholesterol. CONCLUSIONS: Our data underscore the potential relevance of PBMC cholesterol in in vivo HIV replication and the complex effects of atorvastatin that seem to be unrelated to PBMC cholesterol.