Unit: Viral Immunology

Director: VIRELIZIER Jean-Louis

The laboratory has been involved for years in the molecular investigation in vivo and in vitro of host-virus relationship. The general aim is to understand viral strategies in order to better fight infectious diseases of great impact in public health such as HIV/AIDS, hepatitis C (HCV) and dengue virus infections. Approaches are focused on interactions between viral envelope proteins and membrane receptors, critical steps for virus capture, fusion and internalisation. Our knowledge on chemokines and chemokine receptors is used to investigate human haematologic and immunologic disorders of inflammatory and genetic nature.Report:

The work described below was performed under the joint direction of Fernando Arenzana-Seisdedos and Jean-Louis Virelizier

Characterization of HIV signalling in primary CD4 T lymphocytes

Signalling capacity of HIV envelope glycoprotein in primary CD4 T lymphocytes. (L. Chakrabarti, K. Balabanian, J. Harriague, B. Lagane) As part of a larger, multi-department project (GPH on HIV/AIDS) , we investigate whether the interaction of HIV-1 envelope glycoprotein gp120 with its receptors on CD4+ T lymphocytes can generate an aberrant set of signal transduction cascades that would contribute to the T cell dysfunction characterisic of HIV-1 infection. We report that the spectrum of signalling responses induced by gp120 through the chemokine receptor CXCR4 paralleled that induced by the natural ligand SDF-1/CXCL12. Gp120 promoted activation of heterotrimeric G proteins and of the major G-dependent pathways, including calcium mobilization, PI3-kinase and Erk-1/2 MAP kinase activation. In addition, gp120 caused rapid actin cytoskeleton rearrangements and profuse membrane ruffling, as evidenced by dynamic confocal imaging (see enclosed photograph). This coordinated set of events resulted in a bona fide chemotactic response. Inactivated HIV-1 virions that harbored conformationally intact envelope glycoproteins did cause actin polymerization and chemotaxis, while similar virions devoid of envelope glycoproteins did not. Thus gp120, in monomeric as well as oligomeric, virion-associated form, elicited a complex cellular response that mimicked the effects of a chemokine. Therefore, HIV-1 has the capacity to attract or retain potential target cells, change their activation status, and locally perturb their cytoskeleton, all of which may promote viral entry and propagation (Balabanian et al., J. Immunol. 2004)

Genomic analysis of SDF-1- or HIV-induced gene expression (F. Arenzana-Seisdedos, E. Cabannes, Klemm, L.)

In collaboration with the Laboratory of Functional Genomic at Evry Genopole, Dr. Xavier Gidrol, and the Université Libre de Bruxelles, Pr. Parmentier). We have identified genes induced or repressed by HIV in purified CD4+ T lymphocytes following occupation by X4 HIV (molecular clone pNL4-3) of the receptor complex. We have followed in parallel the transcriptional profile induced upon interaction of the natural ligand SDF-1 with CXCR4. We developed a cDNA microarray based on more than 10000 non-redundant clones enriched in selectively expressed lymphocyte genes. Experiments were performed in leukocytes from 5 healthy individuals (representing a total of 400 arrays), using the dye-swap approach. Our microarray data were validated by quantitative PCR. Statistic analysis was performed using a particularly stringent method (Bonferroni analysis). Biological results and clustering data are now being analysed.

Role of cell membrane lectin receptors (DC-SIGN / L-SIGN) expression in hepatitis C virus infection (Lozach, P-Y, A. Amara , R. Altmeyer)

We previously showed that the C-Type lectin DC-SIGN is involved in infection and dissemination of human cytomegalovirus (Halary et al, 2002,. Immunity 17, 653-664. ) and dengue virus ( Navarro-Sanchez et al, 2003, ..Embo R. , 4 , 7 , :723-8 .). The molecular mechanisms involved in the hepatic tropism of hepatitis C virus (HCV) have not been identified. We previously showed that liver-expressed C-type lectins L-SIGN and DC-SIGN bind the HCV E2 glycoprotein with high affinity ( Lozach, P. Y et al, (2003) J. Biol. Chem. 278, 20358-20366) . To analyze the functional relevance of this interaction, we generated pseudotyped lentivirus particles presenting HCV glycoproteins E1 and E2 at the virion surface (HCV-pp). High mannose N-glycans are present on E1 and, to a lesser extent, on E2 proteins of mature infectious HCV-pp. Such particles bind to both L-SIGN and DC-SIGN, but they cannot use these receptors for entry into cells. However, infectious virus is transmitted efficiently when permissive Huh-7 cells are cocultured with HCV-pp bound to L-SIGN or to DC-SIGN-positive cell lines. HCV-pp transmission via L-SIGN or DC-SIGN is inhibited by characteristic inhibitors such as the calcium chelator EGTA and monoclonal antibodies directed against lectin carbohydrate recognition domains of both lectins. In support of the biological relevance of this phenomenon, dendritic cells expressing endogenous DC-SIGN transmitted HCV-pp with high efficiency in a DC-SIGN-dependent manner. Our results support the hypothesis that C-type lectins such as the liver sinusoidal endothelial cell-expressed L-SIGN could act as a capture receptor for HCV in the liver and transmit infectious virions to neighboring hepatocytes.(Lozach PY, Amara A, Bartosch B, Virelizier JL, Arenzana-Seisdedos F, Cosset FL, Altmeyer R., J. Biol. Chem. 2004 Jul 30;279 (31):32035-45)

Physiopathology of SDF-1/CXCR4 signalling (F. Bachelerie, K. Balabanian, B. Laganne, T. Planchenault)

In collaboration with the French National Register of Neutropenias and a network of major French hospitals , we investigated the molecular and genetic basis of the rare, inherited WHIM syndrome. This clinically orientated, although highly fundamental approach of our laboratory complies with the aims and tasks of the department of Molecular Medicine, to which our laboratory is integrated, and of which the head of unit is director (see the site : http://www.pasteur.fr/recherche/departements/DepMedmol/). In collaboration with the National Register of Neutropenias and a number of major French hospitals involved in the diagnosis and treatment of febrile neutropenias we have identified recorded and investigated in the last 2 years 13 cases of this rare pathology. This work enabled the laboratory to be appointed by the European Society of Immunodeficiencies as a laboratory reference for the study of chemotactic disorders of leukocytes.

The WHIM syndrome is characterized by high susceptibility to HPV infections (Warts), Hypogammaglobulinemia (H), recurrent bacterial infections (I) and retention of mature neutrophils or myelokathexis in the bone marrow (M). Dominant heterozygous mutations of the gene encoding CXCR4, a G protein-coupled receptor with a unique ligand, CXCL12, have been associated with this pathology. We studied patients belonging to three different pedigrees. Two siblings inherited a CXCR4 mutation encoding a novel C-terminally truncated receptor while two unrelated patients were found to bear a wild-type CXCR4 open reading frame. Circulating lymphocytes and neutrophils from all patients displayed similar functional alterations of CXCR4-mediated responses featured by a marked enhancement of G-protein dependent responses. We showed that this enhanced response results from the refractoriness of CXCR4 to be both desensitized and internalized in response to CXCL12. Therefore, the aberrant dysfunction of the CXCR4-mediated signaling constitutes a common biological trait of WHIM syndromes , as a result of different genetic anomalies. Responses to other chemokines, namely CCL4, CCL5 and CCL21, were preserved, suggesting that in clinical forms associated with a wild-type CXCR4 open reading frame, the genetic anomaly might target an effector with some degree of selectivity for the CXCL12/CXCR4 axis. We propose that the sustained CXCR4 activity in patient cells accounts for the immune-haematological clinical manifestations and the profusion of warts characteristic of the WHIM syndrome (Balabanian et al., Blood, 2004).

Legend of illustration : Polymerisation of actin skeleton induced by HIV gp120 enveloppe protein (200nM, I min) in primary CD4 T lymphocytes showing : in left panel , polymerised actin (red, phalloidin-FITC)(blue = DAPI stain, nucleus), no stain of the same cell (central panel), and (right panel) a 3D reconstitution of 2 different lymphocytes stained as in the left panel. Keywords : HIV, dengue virus, DC-SIGN, CXCR4, WHIM syndrome

Keywords: HIV, dengue virus, DC-SIGN, CXCR4, WHIM syndrome


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