Our laboratory has a long-standing interest in the study of the molecular aspects of host/HIV relationship. The aim of our research efforts is to better understand the peculiar strategy of indefinite persistence of this lentivirus, in order to fight HIV infection in a manner as adapted as possible to this strategy. Our approaches combine work on transcription of the integrated HIV genome, , as controled through the promoter (LTR) by the transcription factor NF-kB, and on HIV entry by fusion with target cell membranes through interactions with CD4 and chemokine receptors (CCR5 and CXCR4).
These research efforts are coordinated by Fernando Arenzana-Seisdedos and Jean-Louis Virelizier.
Physiopathological aspects of SDF-1 and CXCR4 expression (theme leader : F. Arenzana-Seisdedos)
We previously showed that the chemokine SDF-1 is the unique natural ligand of the membrane receptor CXCR4, one of the main HIV coreceptor (Nature, 1996), and that blockade of CXCR4-using HIV viruses (X4 strains) entry by SDF-1is due to receptor internalisation (J.Exp.Med., 1998).
SDF-1 and mucosal HIV contamination
We have now analysed tissue expression of SDF-1 and its consequences on membrane CXCR4 expression. It was shown that constitutive SDF-1 production in epithelial cells of the human mucosa is associated with down-regulation of CXCR4 on intra-epithelial T lymphocytes (IEL) investigated ex vivo (Agace et al, Current Biology, 2000). The permanent internalisation of CXCR4 in IEL observed in vivo is spontaneously reversed as soon as IEL are cultured at 37°c in vitro. Thus SDF-1 produced in the mucosal environment is likely to down-regulate CXCR4, but not CCR5, expression in T lymphocytes directly encountered by HIV during mucosal transmission, which provides an intriguing, possible explanation of the almost exclusive, venereal contamination by R5, but not X4 viruses. This observation raised the question of whether SDF-1 is secreted as a soluble, diffusible chemokine, or is maintained locally to perform CXCR4 activation through direct cell/ cell contact between epithelial cells and IEL. We previously showed that SDF-1 is exposed in a potentially active manner on the cell membrane through interaction between a defined domain of its C-terminal and membrane heparan sulfates (Amara et al, J.Biol.Chem, 1999), the latter being expressed on the laterobasal side of mucosal epithelial cells, where IEL are also situated. We now have shown that optimal inhibition of X4 HIV isolates by SDF-1 requires interaction with cell-surface heparan sulfates (Valenzuela-Fernandez et al, J.Biol. Chem., 2001). Altogether these results are compatible with the hypothesis that locally secreted, membrane-bound SDF-1 participates in host defense against venereal HIV X4 contamination by constitutively down-regulating CXCR4 on CD4 target cells of the mucosa.
1)Role of SDF-1 in the physiology of the immune system and hematopoiesis , and in the pathogenesis of inflammatory arthritis
In order to perform their many functions in normal or pathological tissues of the body, circulating leukocytes must leave the lumen of blood vessel and find their way through a continuous barrier of endothelial cells (extravasation or diapedesis). We have shown that membrane expression of SDF-1 on human endothelial cells is sufficient for induction of CXCR4-expressing leucocyte tethering and rolling on VCAM-1 under flow conditions in vitro (Grabovsky et al, J.Exp. Med., 2000). Repopulation of murine bone-marrow by precursor cells after irradiation was also shown to result from induction of SDF-1 expression following DNA damage (Ponomaryov et al, J.Clin. Invest., 2000). In addition to epithelial and endothelial cells, mesothelial cells also constitutively produce SDF-1. Peritoneal cells of mesothelial lineage were shown to have a major role in recruting and activating B lymphocytes in the murine peritoneum ( Foussat et al, Eur.J. Immunol., 2001). Aberrant recrutment and activation of leukocytes in tissues participates in the process of inflamation. We found SDF-1 on synovial endothelial cells and showed that SDF-1 is able to induce strong integrin-mediated adhesion of synovial fluid T lymphocytes to fibronectin and ICAM-1, when TGF-beta induces CXCR4 on these lymphocytes ( Buckley et al, J.Immunol., 2000).
2) Interactions of HIV envelope with CD4 and coreceptors, and the fusion phenomenon (theme leader : Ralf Altmeyer)
The HIV envelope protein interacts successively with CD4 and either CCR5 or CXCR4, thus inducing HIV fusion and entry. The envelope protein is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. Using cotransfection of the furin protease, we could purified secreted forms of the envelope protein deleted of intracytoplasmic domain (gp140) as mono-, di- and trimers. We observed that trimers show greatly reduced binding to CD4, and neither monomers nor trimers bound directly to CCR5 (Straropoli et al, J.Biol. Chem., 2000). This implies that gp120/gp41 oligomers on the virion surface are stabilized by a mechanism which remains to be identified. We are presently developping a quantitative fusion assay using cytofluorography to answer these questions, and analyse the ability of envelope expressing cells to fuse with resting T lymphocytes. We also used these materials and methods to show a direct role of membrane-expressed gp120/gp41 in the toxicity to human astrocytes induced by HIV-infected cells (Boutet et al, AIDS, 2000). A differential signalling of SDF-1 and HIV glycoprotein was observed in rat neurons and astrocytes (Lazarini et al, Eur. J. Neuroscience, 2000).
3) Regulation of NF-kB function in the nucleus through inducible, intranuclear IkB alpha degradation by the proteasome (theme leader : F. Bachelerie)
For optimal induction of transcriptional activity of the HIV-LTR and many cellular genes, the transcription factor NF-kB must remain active in the nucleus for many hours after its translocation into the nucleus. However, NF-kB nuclear activity is almost immediately terminated by its own ability to induce neotranscription and synthesis of the inhibitor IkB-alpha, which translocates into the nucleus and dissociates NF-kB from its bound DNA sequences, as we reported previously. We now showed that inducers of NF-kB such as TNF induce ubiquitination and proteasome-dependent degradation of intranuclear IkB, thus permiting persistent NF-kB activity as long as stimulation is maintained ( Renard et al, J. Biol. Chem., 2000).
Photo: Cells fused (yellow) from CD4/coreceptor-expressing cells (green) and HIV envelope expressing cells (red)