Lucile Espert

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.

Involvement of autophagy in viral infections: antiviral function and subversion by viruses

Autophagy is a cellular process involved in the degradation and turn-over of long-lived proteins and organelles, which can be subjected to suppression or further induction in response to different stimuli. According to its essential role in cellular homeostasis, autophagy has been implicated in several pathologies including cancer, neurodegeneration and myopathies. More recently, autophagy has been described as a mechanism of both innate and adaptive immunity against intracellular bacteria and viruses. In this context, autophagy has been proposed as a protective mechanism against viral infection by degrading the pathogens into autolysosomes. This is strengthened by the fact that several proteins involved in interferon (IFN) signalling pathways are linked to autophagy regulation. However, several viruses have evolved strategies to divert IFN-mediated pathways and autophagy to their own benefit. This review provides an overview of the autophagic process and its involvement in the infection by different viral pathogens and of the connections existing between autophagy and proteins involved in IFN signalling pathways.

Interferons alpha and gamma induce p53-dependent and p53-independent apoptosis, respectively

Type I interferon (IFN) enhances the transcription of the tumor suppressor gene p53. To elucidate the molecular mechanism mediating IFN-induced apoptosis, we analysed programmed cell death in response to type I (IFNα) or type II (IFNγ) treatment in relation to p53 status. In two cell lines (MCF-7, SKNSH), IFNα, but not IFNγ, enhanced apoptosis in a p53-dependent manner. Furthermore, only IFNα upregulated p53 as well as p53 target genes (Noxa, Mdm2 and CD95). The apoptotic response to IFNα decreased in the presence of ZB4, an anti-CD95 antibody, suggesting that CD95 is involved in this process. When p53 was inactivated by the E6 viral protein or the expression of a p53 mutant, IFNα-induced apoptosis and p53 target genes upregulation were abrogated. Altogether these results demonstrate that p53 plays a pivotal role in the IFNα-induced apoptotic response. IFNα-induced PML was unable to recruit p53 into nuclear bodies and its downregulation by siRNA did not alter CD95 expression. In contrast, IFNγ-induced apoptosis is p53-independent. CD95 and IFN-regulatory factor 1 (IRF1) are directly upregulated by this cytokine. Apoptotic response to IFNγ is decreased in the presence of ZB4 and strongly diminished by IRF1 siRNA, implicating both CD95 and IRF1 in IFNγ-induced apoptotic response. Taken together, these results show that in two different cell lines, IFNα and IFNγ, induce p53-dependent -independent apoptosis, respectively.

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.

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.

Hyaluronan, a major non‐protein glycosaminoglycan component of the extracellular matrix in human bone marrow, mediates dexamethasone resistance in multiple myeloma

Summary. Originating from a post‐switch memory B cell or plasma cell compartment in peripheral lymphoid tissues, malignant multiple myeloma (MM) cells accumulate in the bone marrow of patients with MM. In this favourable microenvironment, their growth and survival are dependent upon both soluble factors and physical cell‐to‐cell and cell‐to‐extracellular‐matrix contacts. In this study, hyaluronan (HA), a major non‐protein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, acted as a survival factor against dexamethasone (Dex)‐induced apoptosis in MM cell lines. These effects were mediated through an interleukin 6 (IL‐6) autocrine pathway, involving signal transducers and activators of transcription‐3 phosphorylation on IL‐6‐dependent XG‐1 and XG‐6 cell lines. HA promoted accumulation of IL‐6 in the culture medium without affecting IL‐6 gene expression, suggesting that HA protects, stabilizes and concentrates IL‐6 close to its site of secretion, thus favouring its autocrine activity. In contrast, in the IL‐6‐independent RPMI8226 cell line, HA survival effect was mediated through a gp80‐IL‐6 receptor‐independent pathway, resulting in the upregulation of Bcl‐2 anti‐apoptotic protein expression and nuclear factor‐κB activation. Taken together, these data suggest that HA antagonizes Dex‐induced apoptosis of MM cells by favouring the autocrine activity of different cytokines or growth factors. As HA is a major component of the bone marrow extracellular matrix, these findings support the idea that HA could play a major role in the survival of MM cells in vivo, and could explain why MM cells accumulate in the bone marrow of patients with MM and escape conventional chemotherapy.

The exonuclease ISG20 mainly localizes in the nucleolus and the Cajal (Coiled) bodies and is associated with nuclear SMN protein-containing complexes.

We have previously shown that ISG20, an interferon (IFN)‐induced gene, encodes a 3′ to 5′ exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single‐stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin‐dsRed) increased the number of nuclear dots containing both ISG20 and coilin‐dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)‐containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN‐mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation. J. Cell. Biochem. 98: 1320–1333, 2006.

The exonuclease ISG20 is directly induced by synthetic dsRNA via NF-κB and IRF1 activation

Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus–host interactions. Recently, we demonstrated that the IFN-induced 3′ → 5′ exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-κB factors p50 and p65 bind and activate the κB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-κB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.