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  Director : Jean-Louis VIRELIZIER (virelizi@pasteur.fr)


  abstract

 

Our laboratory concentrates its approaches on HIV interactions with host cell membranes, to better understand the strategy of persistence of the virus, and some unsolved aspects of HIV infection : early contamination and dissemination, escape from chemokine control, usage of more membrane structures than previously thought (each being potential target for compounds blocking these interactions), HIV entry in resting cells used as reservoirs, and induction by HIV envelope of abnormal T cell differentiation states.



  report

cale

The coordination of our research efforts was made jointly by Fernando Arenzana-Seisdedos and Jean-Louis Virelizier

1. Post-translational regulation of SDF1 -1 and CXCR4 expression - Responsible researchers : Fernando Arenzana-Seisdedos, Agustin. Valenzuela , Thierry Planchenaut, and Isabelle Straropoli. - Collaboration: F.Baleux ( IP ); Chignard ( IP), H.Lortat-Jacob (Grenoble).

Among the many known chemokines, most are transcribed de novo upon active induction, whereas a few others are produced constitutively. In the case of the former, production is likely to cease naturally, when induction is finished. In the latter( such as SDF-1), however, production and activity is permanent. It may thus be postulated that some kind of post-translational mechanism terminates, when it is not any more needed, either chemokine activity or receptor function

Our laboratory previously reported (EMBO J., 1997) that deletion of as few as the 2 last N-terminal aa in the SDF-1 molecule abolishes chemotactic and HIV entry blocking properties. Similarly, the extracellular N-terminus end of CXCR4 is known to be required for high affinity binding of the chemokine. We have launched an investigation to biochemically and functionally characterize the changes induced by limited proteolysis of the chemokine SDF-1 and its ligand CXCR4 in a biologically relevant environment, using supernatants of resting or activated PBL, purified T lymphocytes or polymorphs. C-terminally biotinylated chemokines were synthesised, and monoclonal antibodies recognising the N-terminal domains were previously developed. Sites of proteolytic cleavage were identified using HPLC purification and mass-spectrometry on the resulting cleavage products. Our results indicate that elastase produced by polymorphs has a major role in terminating the functions of both SDF-1 and CXCR4, through specific cleavage of the N-terminal end of each molecule.

2. Spatial redistribution of molecules involved in HIV/cell membrane interactions and signalling , inside and outside microdomains -Responsible researchers : Francoise Bachelerie, Yann Percherencier, Thierry Planchenault . Collaboration : Hoessli (Centre médical Universitaire, Geneva), J-L Galzi (Strasbourg), A. Lopez (IPBS/CNRS, UMR 5089, Toulouse).

The plasma membrane is not any more seen as a neutral two-dimensional solvent. It is now accepted that there are lateral regionalization of membrane components which can be described in terms of domains. Recent advances lead to the identification of lateral assemblies of sphingolipids and cholesterol or rafts as platforms to support numerous cellular functions, including sorting of proteins involved in signal transduction processes, trafficking of proteins from the trans-golgi network and virus entry or budding. However, at present the relationship between the cell signaling pathways induced by the binding of the HIV Env and the fate of viral cycle is not known. Work carried out in our laboratory clearly indicates that HIV infection is independent of G-protein signaling, since it occurs in CD4 T lymphocytes expressing a CCR5 cDNA containing mutations of the DRY domain abolishing G-protein-dependent signals (in preparation). We have investigated some of the molecular mechanisms controlling the lateral distribution of CD4 and HIV-coreceptors in membranes, as well as their functional significance for viral attachment, membrane fusion, and HIV-mediated signaling events. We have observed that palmitoylation of CCR5 occurs on three cysteine residues in its cytoplasmic tail. Selective mutation of these cysteine residues resulted in a profound decrease in CCR5 membrane expression due to accumulation of the palmitoylation-deficient CCR5 in lysosomal compartments, and its early degradation by a proteasome-independent process (Percherancier et al, J. Biol. Chem., 2001).

3. Conformation requirements for gp120-dependent HIV fusion into resting T lymphocytes Responsible researchers : Ralf Altmeyer, Pierre-Yves Lozach, Chantal Chanel, Brunilde Gril, Isabelle Staropoli

While cellular activation is necessay for productive infection of CD4+ T cells, it remains controversial whether viral entry occurs in resting CD4+ T cells. Entry of HIV into target cells requires the interaction between the viral envelope glycoproteins gp120/gp41 (Env) and cellular receptors CD4 and chemokine receptors CCR5 or CXCR4. Depending on the use of these chemokine receptors HIV strains are divided into R5 and X4 strains respectively. We asked the question whether R5 and X4 strains were able to enter resting CD4+ T cells and to what extent entry correlates with co-receptor expression levels. To this end we used the fluorescence-based envelope-triggered cell-cell fusion assay (FLASH) allowing to directly quantify syncytia formation with primary cells. When Env-expressing BHK cells are co-cultured with monocyte-depleted unstimulated PBMC isolated from four different donors, fusion analysis reveals a donor-dependent variablity of 2 to 3 fold. Bead-purified, resting CD4+ T cells, as well as naive (CD45RA+) and memory (CD45RA-) cells were tested in co-culture with cells expressing the X4-envelope NL4-3 or the R5-envelope BX08. EnvBX08-mediated fusion was inhibited by the CCR5 antagonist TAK779.. Our results indicate, paradoxically, that co-receptor expression does not correlate with fusogenicity: While CXCR4 is readily detected on 97% of cells, CCR5 expression is undetectable on 99% of CD4+ T cells. We could exclude that the EnvBX08 used a coreceptor other than CCR5 as CD4+ T cells from a donor carrying the CCR5*32/*32 deletion did not fuse with EnvBX08, but readily fused with EnvX4. These results indicate the capacity of a R5 envelope to exploit very low levels of CCR5 in primary resting CD4+ T cells, and suggest a novel mechanism used by HIV to create latent cell reservoirs in non permissive T cells.

4 . Creation of DNA microarrays, analysis of novel gene libraries, and test of lymphocyte responses to SDF-1 and HIV . Responsible researchers: F. Arenzana-Seisdedos, Eric Cabannes, Lysiane Laurent. Collaboration: CEA genopole (Evry, France)

This work is based on an interactive collaboration permitting to associate the high technologies developed in the Evry genopole (robots, bioinformatics, design and use of original microarrays), and our laboratory (bringing expertise in relevant T cell systems, chemokine signalling, HIV manipulation in P3 laboratory, etc…). The approach chosen associates microarray analysis and the sensitive SSH (suppression substractive hybridisation) technique, a combination of complementary methods. We use an original DNA library of activated PBLs, presently being sequenced, to be added to that of 16.000 non-redundant clones (French National Sequencing Center). Our present data identified genes induced by SDF-1 in circulating leukocytes, and are being extended to HIV particle-mediated gene induction.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Goupil Marie-Laure goupil@pasteur.fr

Arenzana-Seisdedos Fernando , INSERM , DR1, farenzan@pasteur.fr

Altmeyer Ralf , I.Pasteur , CR , altmeyer@pasteur.fr

Amara Ali, INSERM , CR1 , aamara@pasteur.fr

Bachelerie Françoise, INSERM , CR1 , fbachele@pasteur.fr

Chakrabarti Lisa, I.Pasteur, CR, chakra@pasteur.fr

Virelizier Jean-Louis, Pr . I. Pasteur,/ DRE INSERM, virelizi@pasteur.fr

Cabannes Eric , post-doc, cabannes@pasteur.fr

Valenzuela Agustin, post-doc , avalenzu@pasteur.fr

Percherancier Yann, PhD , ypercher@pasteur.fr

Staropoli Isabelle, engineer , IP, istaro@pasteur.fr

Laurent Lysiane, technician, IP, lyslaure@pasteur.fr

Planchenault Thierry, technician, IP, tplanche@pasteur.fr


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