Marta Curriu

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.

Screening NK-, B- and T-cell phenotype and function in patients suffering from Chronic Fatigue Syndrome

BackgroundChronic Fatigue Syndrome (CFS) is a debilitating neuro-immune disorder of unknown etiology diagnosed by an array of clinical manifestations. Although several immunological abnormalities have been described in CFS, their heterogeneity has limited diagnostic applicability.MethodsImmunological features of CFS were screened in 22 CFS diagnosed individuals fulfilling Fukuda criteria and 30 control healthy individuals. Peripheral blood T, B and NK cell function and phenotype were analyzed by flow cytometry in both groups.ResultsCFS diagnosed individuals showed similar absolute numbers of T, B and NK cells, with minor differences in the percentage of CD4+ and CD8+ T cells. B cells showed similar subset frequencies and proliferative responses between groups. Conversely, significant differences were observed in T cell subsets. CFS individuals showed increased levels of T regulatory cells (CD25+/FOXP3+) CD4 T cells, and lower proliferative responses in vitro and in vivo. Moreover, CD8 T cells from the CFS group showed significantly lower activation and frequency of effector memory cells. No clear signs of T-cell immunosenescence were observed. NK cells from CFS individuals displayed higher expression of NKp46 and CD69 but lower expression of CD25 in all NK subsets defined. Overall, T cell and NK cell features clearly clustered CFS individuals.ConclusionsOur findings suggest that alterations in T-cell phenotype and proliferative response along with the specific signature of NK cell phenotype may be useful to identify CFS individuals. The striking down modulation of T cell mediated immunity may help to understand intercurrent viral infections in CFS.

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

BackgroundCell-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.ResultsThe 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 α and β 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.ConclusionIn 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.

Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

BackgroundThe MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals.We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences.ResultsMiniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses.ConclusionsAnti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses.

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy, Viral Evolution and Compartmentalization

Background Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain. Methods & Results We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized. Conclusions The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

Background CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. Methods We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. Results Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. Conclusions Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41.

Assessing main death pathways in T lymphocytes from HIV infected individuals.

Increased lymphocyte death is a hallmark of human immunodeficiency virus (HIV) infection. Although virological factors have been linked to this phenomenon, increased cell death rates are still observed in treated individuals in which viral replication is halted. To understand the nature of this remaining altered cell death, we have developed a simple and fast assay to assess major cell death pathways in lymphocytes isolated from HIV‐infected individuals. The combination of three factors: (i) antibody staining to identify CD3+CD4+ and CD3+CD8+ cells, (ii) assessment of mitochondrial and plasma membrane function using DiOC6(3) or JC‐1 probes and vital dyes, and (iii) caspase inhibition, allowed for the quantification of caspase‐independent and ‐dependent cell death in CD4 and CD8 T cells. The latter mechanism was divided in intrinsic and extrinsic apoptotic pathways according to the sensitivity of the dissipation of mitochondrial membrane potential to Z‐VAD‐fmk or Q‐VD‐oPH treatment. Our data show similar results for both caspase inhibitors in treated infected individuals, whereas Q‐VD‐oPH showed a more potent inhibition in viremic individuals, yielding lower levels of intrinsic apoptosis. Comparison of DiOC6(3) and JC‐1 probes yielded similar results in CD4 T cells, allowing for a clear definition of death mechanism in these cells. However, in CD8 T‐cells, JC‐1 showed heterogeneous staining and detected significantly lower levels of cell death with a higher contribution of intrinsic apoptosis. In conclusion, we provide a simple method to assess CD4 T‐cell death mechanisms in HIV‐infected individuals. The reasons and consequences of mitochondrial heterogeneity in CD8 T‐cells require further evaluation.

The HR2 polymorphism N140I in the HIV-1 gp41 combined with the HR1 V38A mutation is associated with a less cytopathic phenotype

BackgroundResistance to the fusion inhibitor enfuvirtide (ENF) is achieved by changes in the gp41 subunit of the HIV envelope glycoprotein (Env). Specific ENF-associated mutational pathways correlate with immunological recovery, even after virological failure, suggesting that the acquisition of ENF resistance alters gp41 pathogenicity. To test this hypothesis, we have characterized the expression, fusion capability, induction of CD4+ T cell loss and single CD4+ T cell death of 48 gp41 proteins derived from three patients displaying different amino acids (N, T or I) at position 140 that developed a V38A mutation after ENF-based treatment.ResultsIn all cases, intra-patient comparison of Env isolated pre- or post-treatment showed comparable values of expression and fusogenic capacity. Furthermore, Env with either N or T at position 140 induced comparable losses of CD4+ T-cells, irrespective of the residue present at position 38. Conversely, Env acquiring the V38A mutation in a 140I background induced a significantly reduced loss of CD4+ T cells and lower single-cell death than did their baseline controls. No altered ability to induce single-cell death was observed in the other clones.ConclusionsOverall, primary gp41 proteins with both V38A and N140I changes showed a reduced ability to induce single cell death and deplete CD4+ T cells, despite maintaining fusion activity. The specificity of this phenotype highlights the relevance of the genetic context to the cytopathic capacity of Env and the role of ENF-resistance mutations in modulating viral pathogenicity in vivo, further supporting the hypothesis that gp41 is a critical mediator of HIV pathogenesis.

Evaluation of the Cytopathicity (Fusion/Hemifusion) of Patient-Derived HIV-1 Envelope Glycoproteins Comparing Two Effector Cell Lines

HIV-1 envelope glycoprotein (Env) is a major determinant of viral pathogenicity. The evaluation of the biological properties of patient-derived envelopes by comparing two effector cell lines (293T and HeLa) is reported. A standard cell-to-cell fusion assay was used to evaluate fusogenicity, whereas a coculture with CD4+ cells was used to evaluate absolute cell loss, single cell death, and hemifusion events. Fusion and absolute cell loss assays showed that Env-expressing 293T and HeLa cells had different fusion efficiencies; fusion was magnified in 293T cells despite a significantly lower cell-surface Env expression. Conversely, gp41-mediated single cell death and hemifusion induced in CD4+ cells by 293T-Env-positive cells were significantly lower than that induced by HeLa-Env-positive cells. These data showed that the effector cell line used in the in vitro assays is crucial, and a combination of assays is recommended to evaluate the biological properties of patient-derived envelope glycoproteins: preferentially, 293T-Env-positive cells for the evaluation of fusogenicity and HeLa-Env-positive cells for the evaluation of cell death parameters. The combination of assays described in our work could be a valuable tool for dual screenings of large collections of primary Envs or Env mutants and drugs acting on these Envs.

Susceptibility of human lymphoid tissue cultured ex vivo to xenotropic murine leukemia virus-related virus (XMRV) infection.

Background: Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored Results: XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. Conclusions: Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus.