Véronique Robert-Hebmann

Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4

HIV-1 envelope glycoproteins (Env), expressed at the cell surface, induce apoptosis of uninfected CD4+ T cells, contributing to the development of AIDS. Here we demonstrate that, independently of HIV replication, transfected or HIV-infected cells that express Env induced autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. The same phenomena occurred in a T cell line and in transfected HEK.293 cells that expressed both wild-type CXCR4 and a truncated form of CD4 that is unable to bind the lymphocyte-specific protein kinase Lck. Env-mediated autophagy is required to trigger CD4+ T cell apoptosis since blockade of autophagy at different steps, by either drugs (3-methyladenine and bafilomycin A1) or siRNAs specific for Beclin 1/Atg6 and Atg7 genes, totally inhibited the apoptotic process. Furthermore, CD4+ T cells still underwent Env-mediated cell death with autophagic features when apoptosis was inhibited. These results suggest that HIV-infected cells can induce autophagy in bystander CD4+ T lymphocytes through contact of Env with CXCR4, leading to apoptotic cell death, a mechanism most likely contributing to immunodeficiency.

Differential role of autophagy in CD4 T cells and macrophages during X4 and R5 HIV-1 infection.

BACKGROUND HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection. CONCLUSIONS/SIGNIFICANCE Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here, we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.

Apoptosis of uninfected cells induced by HIV envelope glycoproteins

Apoptosis, or programmed cell death, is a key event in biologic homeostasis but is also involved in the pathogenesis of many human diseases including human immunodeficiency virus (HIV) infection. Although multiple mechanisms contribute to the gradual T cell decline that occurs in HIV-infected patients, programmed cell death of uninfected bystander T lymphocytes, including CD4+ and CD8+ T cells, is an important event leading to immunodeficiency. The HIV envelope glycoproteins (Env) play a crucial role in transducing this apoptotic signal after binding to its receptors, the CD4 molecule and a coreceptor, essentially CCR5 and CXCR4. Depending on Env presentation, the receptor involved and the complexity of target cell contact, apoptosis induction is related to death receptor and/or mitochondria-dependent pathways. This review summarizes current knowledge of Env-mediated cell death leading to T cell depletion and clinical complications and covers the sometimes conflicting studies that address the possible mechanisms of T cell death.

The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies

BackgroundThe human T-cell leukemia virus type I (HTLV-I) basic leucine-zipper factor (HBZ) has previously been shown to modulate transcriptional activity of Jun family members. The presence of a novel isoform of HBZ, termed HBZ-SP1, has recently been characterized in adult T-cell leukemia (ATL) cells and has been found to be associated with intense nuclear spots. In this study, we investigated the role of these nuclear bodies in the regulation of the transcriptional activity of JunB.ResultsUsing fluorescence microscopy, we found that the HBZ-SP1 protein localizes to intense dots corresponding to HBZ-NBs and to nucleoli. We analyzed the relative mobility of the EGFP-HBZ-SP1 fusion protein using fluorescence recovery after photobleaching (FRAP) analysis and found that the deletion of the ZIP domain perturbs the association of the HBZ-SP1 protein to the HBZ-NBs. These data suggested that HBZ needs cellular partners, including bZIP factors, to form HBZ-NBs. Indeed, by cotransfection experiments in COS cells, we have found that the bZIP factor JunB is able to target delocalized form of HBZ (deleted in its nuclear localization subdomains) into the HBZ-NBs. We also show that the viral protein is able to entail a redistribution of JunB into the HBZ-NBs. Moreover, by transfecting HeLa cells (known to express high level of JunB) with a vector expressing HBZ-SP1, the sequestration of JunB to the HBZ-NBs inhibited its transcriptional activity. Lastly, we analyzed the nuclear distribution of HBZ-SP1 in the presence of JunD, a Jun family member known to be activated by HBZ. In this case, no NBs were detected and the HBZ-SP1 protein was diffusely distributed throughout the nucleoplasm.ConclusionOur results suggest that HBZ-mediated sequestration of JunB to the HBZ-NBs may be causing the repression of JunB activity in vivo.

Identification of the Cytoplasmic Domains of CXCR4 Involved in Jak2 and STAT3 Phosphorylation

The chemokine SDF-1α transduces Gi-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a Gi-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1α binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr135 in the DRY motif at the N terminus of ICL2 nor the Tyr65 and Tyr76 in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1α binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.

Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat in CD4+ T Lymphocytes

ABSTRACT Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4+ T lymphocytes, a mechanism likely contributing to the loss of CD4+ T cells. In contrast, in productively infected CD4+ T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4+ T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread. IMPORTANCE Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4+ T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

Clonal analysis of murine b cell response to the human immunodeficiency virus type 1 (HIV1)-gag p17 and p25 antigens

The antigenicity of HIV-gag p17 and p25 proteins was analyzed using a panel of 52 monoclonal antibodies (mAb) derived from 17 independent fusion experiment protocols performed in 12 different laboratories. These mAb were tested for their capacity to bind peptides corresponding to sequences of HIV1-BRU-gag p17 and p25. Thirty-five overlapping peptides (P1 to P35) totally covering the p17 and p25 proteins were used. This study allowed us to identify four immunodominant regions inducing B cell response, two on p17 corresponding to P2 and P13 (amino acids 11-25 and 121-132, respectively) and two on p25 corresponding to P21 and P28-P29-P30 (a.a. 201-218 and 285-320 respectively). According to secondary structure predictions, peptides P2 and P21 contained hydrophilic alpha helix folded regions whereas P13 sequence presented a beta turn propensity. These regions and the P28-30 region were also predicted to be easily accessible to mAb. Several other p25-derived peptides: P15 (a.a. 142-156), P16 (a.a. 148-162), P19 (a.a. 176-192), P22 (a.a. 219-233) and P23 (a.a. 233-253) were recognized by mAb. No p17-derived peptide other than P2, P13 and P12 (a.a. 111-123) was found to react with mAb. Cross-blocking studies between mAb, suggested the existence of more than four distinct epitopic areas on p17 and eight on p25.

The lck protein tyrosine kinase is not involved in antibody‐mediated CD4 (CDR3‐loop) signal transduction that inhibits HIV‐1 transcription

Monoclonal antibodies (mAb) that bind to the immunoglobulin CDR3‐like region in the D1 domain of the CD4 molecule can inhibit the HIV‐1 life cycle in CD4‐positive T cells and lymphoblastoid cell lines at the stage of transcription. This antiviral effect requires the integrity of the cytoplasmic tail of CD4 which is known to act as a signal transduction region through its association with the protein tyrosine kinase (PTK) p56lck. In this study, we investigated the putative role of this PTK in transducing inhibitory signals that act on HIV‐1 replication after triggering by anti‐CDR3‐like region antibody treatment of infected T cell lines. CEM (CD4+/p56lck + inducible), MT2 (CD4+/p56lck – repressed), HSB‐2 (CD4–/p56lck + constitutively), HSB‐2 WTCD4 (CD4+/p56lck + constitutively), HSB‐2 CD4.402 (CD4+ truncated form which lacks the cytoplasmic domain/p56lck + constitut ively), and HSB‐2 CD4mut (CD4+ unable to bind lck/p56lck + constitutively) were exposed to HIV‐1 and cultured in medium supplemented with an anti‐CDR3‐like region‐specific antibody or a control anti‐CD4 mAb which does not inhibit HIV‐1 transcription. We found that CDR3‐loop‐mediated inhibitory signals are efficiently transduced in CD4‐positive cells which demonstrate a constitutive activation of p56lck or in CD4‐positive cells lacking p56lck expression. Moreover, inhibitory signals were transduced in HSB‐2 CD4mut cells expressing a cell surface CD4 with a double cysteine mutation in its cytoplasmic tail that renders the molecule unable to bind p56lck, but not HSB‐2 CD4.402 cells expressing a truncated form of CD4 which lacks the cytoplasmic domain. These results indicate that the p56lck plays no direct role in this process and suggests the existence of another signaling partner for CD4.

Autophagy plays an essential role in HIV-1 infection

HIV-1 can infect and replicate in both CD4 T cells and macrophages, and direct cell-to-cell spread is an important route of HIV-1 propagation. It requires interaction between HIV-1 envelope glycoproteins (Env, composed of gp120 and gp41), expressed at the surface of infected cells, and HIV-1 receptors, CD4 and a coreceptor, on the target cells. The gp120 interacts first with CD4, which triggers conformational changes leading to increased exposure of gp120 regions (including the V3 loop) able to bind to the coreceptor, mainly CCR5 or CXCR4. This interaction induces a structural rearrangement in gp41, insertion of its N terminus fusion peptide into the target membrane, and fusion. R5 and X4 HIV-1 strains use CCR5 and CXCR4, respectively, for entry. We have previously demonstrated that, independently of HIV-1 replication, X4 HIV-infected cells trigger autophagy in the uninfected CD4 T lymphocytes. Env-mediated autophagy is dependent on the gp41 fusogenic activity but is independent of a direct CD4- and CXCR4-mediated signaling pathway. Furthermore, this autophagy process is required to trigger CD4 T cell apoptosis since blockade of autophagy at different steps, by either drugs or short interfering RNAs specific for autophagy genes, totally inhibits Env-mediated apoptosis. Our last results show that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, two populations of autophagic macrophages can be observed during their coculture with HIV-1-infected cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection. Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.