|PDF Version||Virology and Cellular Immunology - URA CNRS 1930|
|Director : Hovanessian, Ara (firstname.lastname@example.org)|
The mechanism of HIV entry in target cells was investigated in order to design specific inhibitors of HIV infection. In addition, HIV infection, like most of other viruses, induces interferon that eventually controls virus infection. In this respect, the major lines of research are: 1) The mechanism of HIV entry and its inhibition by the pseudopeptide HB-19 and the cytokine Midkine; 2) Role of the virus-activated transcription factor IRF7 in the onset of the innate antiviral response mediated by interferon.
The Anti-HIV Pseudopeptide HB-19 Binds the C-terminal End of Nucleolin and Prevents Anchorage of Virus Particles on Target Cells (A. Hovanessian, B. Krust, E. Said)
The pseudopeptide 5[Ky (CH2N)PR]-TASP, referred to as HB-19, is a potent inhibitor of HIV infection in different CD4+ cell lines as well as in primary T lymphocytes and macrophages. HB-19 binds specifically the cell-surface-expressed nucleolin and blocks virus particle attachment on permissive cells. The binding of HB-19 to the surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. Consistent with these results, the full-length nucleolin was found to bind HB-19 whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, as the domain that binds HB-19. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV attachment.
Anchorage of HIV on Permissive Cells Leads to the aggregation of Viral Particles with Surface Nucleolin at Membrane Raft Microdomains (A. Hovanessian, B. Krust, and J. Svab)
The cross-linking of HIV particles on permissive cells results aggregation of HIV and its colocalization with clustered surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin co-aggregate with glycolipid-enriched membrane-microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEMs-associated proteins. Our findings demonstrate that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process.
Inhibition of HIV Infection by the Physiological Ligand of Nucleolin, the Cytokine Midkine (A. Hovanessian, E. Said, B. Krust, J. Svab)
We have recently shown that the growth factor midkine (MK), implicated in differentiation and development, inhibits HIV infection by a mechanism similar to that of the anti-HIV pseudopeptide HB-19. MK binds specifically surface nucleolin as a low affinity receptor and inhibits HIV attachment to target cells. The binding of MK to surface nucleolin is independent of heparan sulfate or chondroitin sulfate proteoglycans. In view of its enhanced expression in response to physiological agents along with its various growth factor effects, MK should be considered as a cytokine with a potential function in homeostasis. As examples for the potential control of HIV infection by MK, we then show that CD4+ T-cell clones expressing constitutively MK resist HIV infection via inhibition of HIV particle attachment to cells. Furthermore, when CD4+ cells are cocultured with MK producing CD4-negative cell clones, they become resistant to HIV infection. Taken together, our results suggest that MK is a cytokine that could regulate HIV infection in an autocrine or paracrine manner and thus be implicated in HIV pathogenesis.
Development of the Innate Immune Antiviral Response Against Virus Infections by the Expression of Transcription Factor IRF7. (A. Hovanessian, H. Collandre, A. Caillaud and I. Marié)
Interferon regulatory factor 7 (IRF7) is an interferon inducible transcription factor required for induction of delayed early interferon alpha genes and the onset of a potent antiviral state. After induction of IRF7 by autocrine interferon, latent IRF7 is activated by virus-induced phosphorylation on serine residues within the C-terminal regulatory domain.
- Mapping of IRF7 phosphorylation sites and identification new target genes induced directly by IRF7.
The critical role of IRF7 in the induction of type I interferon genes is now well established, however it is important to determine whether IRF7 is also involved in the transcriptional activation of other genes, in particular cytokines and chemokines. Virus-triggered phosphorylation of IRF7 on serine residues is a prerequisite to IRF7 activation. In view of this, we mapped the serine residues target of phosphorylation and designed a constitutively active phosphomimetic mutant by replacing these serines by aspartic acids. We are currently constructing a retroviral vector expressing the wild type and the constitutively active mutant of IRF7 to be used in cells that do not respond to interferon (cells deficient in STAT1 expression) in order to identify new target genes induced directly by IRF7. These retroviral vectors will also be used in T lymphocytes during infection by HIV-1 in an attempt to control virus infection by the production of inerferon. Finally, such retroviral vectors expressing the wild type and the constitutively active mutant of IRF7 have the potential capacity to boost the innate immune response in individuals infected with HIV or with other viruses.
- Acetylation of IRF7 impairs its DNA binding capacity.
In addition to its virus-dependent activation of IRF7 by phosphorylation of the C-terminal serine residues, we have shown that the activity of IRF7 can be modulated in vivo by the histone acetyltransferases PCAF and GCN5. We mapped the acetylation site to a unique lysine residue located in the DNA binding domain of IRF7 at position 92. We showed that a mutant that is not acetylatable by PCAF due to a change in the surrounding amino acid context of lysine 92, shows increased DNA binding and activity compared to wild type IRF7. Conversely, we show that acetylated IRF7 displays impaired DNA binding capability and that overexpression of PCAF leads to decreased IRF7 activity. Together, our results strongly suggest that acetylation of Lysine 92 negatively modulates IRF7 DNA binding.
Keywords: HIV, HIV entry, pseudopeptide, midkine, interfgeron, IRF7, acetylation
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|Hovanessian, Ara (email@example.com)||HOVANESSIAN Ara, DR I, CNRS/Head of Unit, IP (firstname.lastname@example.org)
KRUST Bernard, INSERM, Researcher (bkrust @pasteur.fr)
MARIE Isabelle, IP, Researcher (email@example.com)
|CAILLAUD Alexandre PhD student (firstname.lastname@example.org)
SAID Elias, PhD student (email@example.com)
|COLLANDRE Hélène, Ingénieur de Recherche I, CNRS (firstname.lastname@example.org)
SVAB Josette, Assistante Ingénieur, CNRS (email@example.com)
FERRIS Stéphane, Technicien Sup., IP (06/02 - 11/02) (firstname.lastname@example.org)
GEORGES, Monique, Aide de laboratoire IP (mi-temps) (email@example.com)
QUEROL, Chantal, Aide de laboratoire IP (mi-temps)