Unit: Viral Immunology

Director: VIRELIZIER Jean-Louis

The laboratory is dedicated to research on host-virus relationship and studies on virus entry, mostly in the HIV but also in the dengue and HCV virus models . The main approaches are : Studies on the signals induced by interaction between viral enveloppe protein through the corresponding membrane receptors (CCR5, CXCR4, CD4, DC-SIGN). Creation of original, human cDNA microarrays to characterise lymphocyte transcriptional responses to HIV. The lectin receptor DC-SIGN , and its role in viral transfer/dissemination. Real-time imaging of early events following cell infection, directly or during transfer by dentritic cells to lymphocytes. Pathological expression/function of CXCR4 in a genetic, immuno-hematological disorder in human (WHIM syndrome).

Changes of distribution, conformation and membrane interactions of CCR5 and CXCR4 upon activation (Francoise Bachelerie, Karl Balabanian, Bernard Lagane, Thierry Planchenaut, Isabelle Staropoli)

These phenomena are investigated in the context of two immune deficiencies : AIDS, and the WHIM syndrome.

Interaction of the HIV envelope protein (env) on CD4 modifies the conformation of env, thus exposing interaction domains of env with chemoreceptors (CCR5 or CXCR4). We previously showed that massive de-localisation of CD4 from lipid microdomains (rafts) to disordered zones of the cell membrane can be induced by appropriate CD4 mutations (Percherancier et al, JBC, 2001). We have now shown that HIV entry is not dependent on CD4 localisation in rafts, but requires membrane cholesterol (Percherancier et al, JBC, 2003).This indicates an important role for cholesterol in maintaining the conformation of the receptor, a critical condition for HIV fusion into target cells. The team now uses dynamic and quantitative imaging approaches (FRET) in living cells to study the regrouping and membrane distribution of viral receptors, in collaboration with biophysicists (A. Lopez and colleagues, Toulouse).

Structure/conformation/function of chemoreceptors in physiology and pathology (WHIM syndrome)

We have observed that CCR5 is expressed in the membrane under different conformation states, depending on its dissociation or constitutive association with G protéins. These observations led us to postulate that some human pathological situations associated with abnormal cell trafficking/migration might be linked to dysfunction of chemoreceptor signalling. The SDF-1/CXCR4 pair controls many physiological functions, including migration of haematopoïetic stem cells to and from the bone marrow .The "WHIM" syndrome, a rare genetic disease, associates Warts, Hypogammaglobulinemia, Infections due to neutropenia, and Myelokathexis (bone marrow retention of myeloid precursors). We have identified in a WHIM family a mutation in the cxcr4 gene resulting in deletion of the last 15 C-ter aa residues. This results in refractoriness of the mutated receptor to homologous desensitisation, leading to a strikingly exagerated chemotactic responses to SDF-1. Interestingly, no cxcr4 mutation, but similar gain of function of CXCR4 responses, were found in another family. This indicates that the biological syndrome responsible for the clinical symptoms of the WHIM syndrome can result from either a genetically mutated cxcr4 gene or other(s) genetic defect affecting CXCR4-dependent signal transduction.

Reversion of a live attenuated SIV vaccine deleted in the nef gene (Lisa Chakrabarti) (collaboration: Cecilia Cheng-Meyer, Aaron-Diamond, NY)

The nef gene is an essential pathogenic determinant of AIDS viruses. Simian immunodeficiency virus deleted in the nef gene (SIVmac239Δnef) causes an attenuated infection with a low viral load, and has been considered as a live attenuated vaccine candidate. However, some of the rhesus macaques inoculated with this attenuated virus show a progressive depletion of CD4+ T lymphocytes and do progress to disease. While studying cases of reversion to pathogenic infections, we showed that the revertant viruses had acquired a second deletion in the nef gene, which resulted in the restoration of the original nef open-reading frame. The resulting protein Nef-Δ2 was defective for several functions such as the induction of MHC class I and CD4 molecules. However, Nef-Δ2 recovered the capacity to promote infection of unstimulated CD4+ T lymphocytes. These data suggest that the revertant protein has recovered the capacity to activate SIV target cells, and thus to promote viral replication. This study shows that a truncated Nef protein can contribute to SIV pathogenesis and underlie potential risks associated to live attenuated vaccines against AIDS.

HIV-1 glycoprotein acts as an agonist of the chemoreceptor CXCR4 : role in acquired immunodeficiency ? (Lisa Chakrabarti, Julie Harriague, Karl Balabanian,Christine Décrion)

We are investigating the role of HIV-1 envelope glycoprotein (Env) in the progressive functional impairment and destruction of CD4+ T lymphocytes that characterizes progression to AIDS. In the process of viral entry, HIV surface glycoprotein gp120 binds to its receptor CD4 and its coreceptor, CXCR4 or CCR5, in a series of steps that lead to a major conformational change of the transmembrane glycoprotein gp41 and to the fusion of viral and cellular membranes. Of note, CD4 and the chemoreceptors CXCR4 and CCR5 are key molecules of the immune system, due to their roles in lymphocyte activation and cellular migration. Gp120 present at the surface of viral particles and infected cells is likely to generate signals through CD4 and the chemoreceptors, which could contribute to the abnormal activation of CD4+ T lymphocytes that is a hallmark of AIDS pathogenesis. We have undertaken to systematically characterize the signaling pathways perturbed by Env in primary CD4+ T lymphocytes, which represent the major target cells of HIV-1.

Gp120 was found to recapitulate the effects of the SDF-1 chemokine, the natural ligand of CXCR4. Migration assays in transwell chambers showed that recombinant gp120LAI induced the chemotaxis of primary CD4+ T lymphocytes, a property that was blocked in presence of the CXCR4 synthetic antagonist AMD3100. Thus, high local concentrations of gp120 may have the capacity to induce the recruitment and / or the retention of HIV target cells. The signaling pathways activated by gp120 parallel those activated by SDF-1, including intracellular calcium mobilization, PI3-kinase activation, induction of actin polymerization, induction of CXCR4 endocytosis, and activation of the MAP kinases Erk 1/2. The kinetics of signal decrease in calcium flux measurements was slower for gp120 than for SDF-1, which suggested differences in the desensitization mechanisms triggered by these two ligands. These data provide evidence for an agonistic effect of gp120 via CXCR4 that may differ from that induced by SDF-1, hence the possibility of an abnormal activation pattern. This project is part of a collaborative program of the Pasteur Institute, the AIDS GPH (Grand Programme Horizontal), that aims at elucidating mechanisms of CD4+ T lymphocyte dysfunction in AIDS.

We are also exploring the signals induced by HIV envelope glycoproteins in the context of interactions between antigen presenting cells and CD4+ T lymphocytes. The re-localization of viral antigens and signaling molecules fused to GFP is studied by dynamic imaging, in collaboration with Spencer Shorte (Dynamic Imaging Center of the Pasteur Institute).

Transcriptional responses of T lymphocytes (human cDNA microarrays) to stimulation by HIV or chemokines. (Eric Cabannes, Lysiane Laurent, Fernando Arenzana). (collaboration: M. Parmentier and colleagues, ULB, Brussels ; X.Gidrol, CEA genopole, Evry; J-Y Coppée, Pasteur microarray station).

There is a need to develop new research tools to investigate, without the usual bias of "candidate genes", the presumably aberrant gene expression pattern induced by HIV infection in human T lymphocytes, in order to investigate the molecular basis of HIV-induced immune disorders. We have developed in house human cDNA microarrays, implying 3 main approaches: 1) Creation of original clone libraries, including genes specifically induced during T cell activation and not represented in available collections 2) Validation of an experimental protocol permitting robust and reproducible results and their statistical analysis (collaboration: J.-J. Daudin, INA P-G, France) .3) Adaptation of this protocol to the conditions imposed by the use of primary T lymphocytes, including unbiased amplification of DNA material , to permit future investigations in rare patient's leukocytes. We are now initiating this transcriptome analysis , and compare transcript expression induced by infectious HIV particles or chemokines (SDF-1 or MIP-1 β ).

Characterisation of a receptor for dengue virus infection of dendritic cells ( Fernando Arenzana, Ralf Altmeyer, Ali Amara) (collaboration :Philippe Despres et al, I. Pasteur) (EMBO Reports 4, 7, 723-728 / 2003)

Dengue virus (DV), a mosquito-borne flavivirus that causes haemorrhagic fever in humans, primarily infects immature dendritic cells (DCs) after a bite by an infected mosquito vector. We have shown that the DC-specific ICAM3-grabbing non-integrin (DC-SIGN) molecule, a cell-surface, mannose-specific, C-type lectin, binds mosquito-cell-derived DVs and allows viral replication in monocyte-derived, human DCs. Conclusive evidence for the involvement of DC-SIGN in DV infection was obtained by the inhibition of viral infection anti-DC-SIGN antibodies or by the soluble tetrameric ectodomain of DC-SIGN. Ectopic, vector-induced DC-SIGN expression in many non DV-permissive cell types induced complete permissiveness to DV replication. Our data show that DC-SIGN functions as a DV-binding lectin by interacting with the DV envelope glycoprotein , provided the latter is appropriately mannosidased. Mosquito-cell-derived DVs may have differential infectivity for DC-SIGN-expressing cells. We suggest that the differential use of DC-SIGN by viral envelope glycoproteins may account for the immunopathogenesis of DV strains . Apart from bringing a long-waited DV receptor characterisation, our results elucidate an intriguing strategy of DV, namely the use of mannosidation of DV envelope by the mosquito vector in order to use a mannose-specific molecule as a receptor in host cells able to replicate and disseminate virus infection .

Keywords: HIV, AIDS,dengue, receptors , dendritic , signals

Activity Reports 2003 - Institut Pasteur

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