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  Director : HOVANESSIAN Ara (arahovan@pasteur.fr)


  abstract

 

In order to clarify the mechanisms implicated in AIDS pathogenesis in relation to HIV infection, the members of this unit have concentrated their studies on the different steps in the HIV infectious cycle, and particularly on the viral entry process. As interferon protects cells against virus infections, studies are carried out in order to understand its mechanism of action. In this respect, the major lines of research are: 1) development of inhibitors of HIV infection, 2) characterization of cell-surface components implicated in the attachment of HIV particles to permissive cells, 3) induction of interferon alpha genes during viral infection , 4) characterization of the different functions of the protein kinase PKR.



  report

cale

The HB-19 pseudopeptide is a potent inhibitor of T-lymphocyte- and macrophage-tropic HIV isolates by blocking the attachment of virus particles to cells (S. Nisole, B. Krust , E. Said, and A.G. Hovanessian )

The HB-19 pseudopeptide that binds the cell-surface expressed nucleolin is a potent inhibitor of HIV infection. By using primary T-lymphocytes and macrophages, and CD4+ cell lines, we showed that HB-19 inhibits in a dose dependent manner both T-lymphocyte- and macrophage-tropic HIV isolates. Similar positively charged control pseudopeptides have no effect on HIV infection even at very high concentrations. These observations and the fact that HB-19 has no effect on HIV-1 pseudotyped with VSV or MoMLV envelope glycoproteins, confirm the specific nature of this inhibitor against the entry process mediated by the HIV envelope glycoproteins. Finally, association of low doses of HB-19 with b
-chemokines or AZT results in an increased inhibitory effect on HIV infection. HB-19 has no inhibitory effect when added to cells after few hours of HIV entry. On the other hand in HB-19 pretreated cells, the inhibitory effect persists for several hours, even after washing cells to remove away the unbound pseudopeptide. Under such conditions, the attachment of HIV particles to cells is inhibited as efficiently as by neutralizing monoclonal antibodies directed against the V3 loop.
The wide spectrum of the inhibitory action of HB-19 against various HIV isolates demonstrates the potential efficacy of this anti-HIV pseudopeptide during the different phases of HIV infection. Moreover, the enhanced inhibitory effect on HIV infection upon association of low doses of HB-19 with b
-chemokines or AZT suggests that simultaneous administration of anti-HIV drugs with HB-19 could provide efficient therapeutic protocols against HIV infection. The wide spectrum of the inhibitory action of HB-19 on different types of HIV isolates, along with its distinct mode of action and stability in serum make this pseudopeptide inhibitor an ideal drug candidate in HIV-infected individuals.


The cell-surface expressed nucleolin is associated with actin microfilaments (A. G. Hovanessian, B. Krust, J. Svab and S. Nisole)

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. On intact cells, cross-linking of the cell-surface-expressed nucleolin with a specific monoclonal antibody, results in its clustering at the external side of the plasma membrane but in close association with intracellular actin. Consequently, disruption of actin microfilaments by cytochalasin D leads to the collapse of clustered nucleolin. Our results confirm that nucleolin is also a component of the cell surface where it could be functional as a cell surface receptor for various ligands including HIV.


Inhibition of HIV Infection by the Cytokine Midkine (S. Nisole, B. Krust and A.G. Hovanessian)

We have recently found that the growth factor midkine (MK), implicated in differentiation and development, binds to the cell-surface expressed nucleolin and results in the inhibition of HIV infection in different cell lines as well as in peripheral blood T-lymphocytes. MK binds cells specifically since this binding is inhibited by unlabeled MK but not by other related growth factors. MK-pretreated cells resist HIV infection by the failure of HIV particles to become attached to cells via a mechanism independent of CD4. We showed that MK mRNA is detectable systematically in adult peripheral blood lymphocytes from healthy donors. Upon activation of T-lymphocytes with PHA or with anti-CD3/anti-CD28 antibodies, the expression of MK is increased significantly in a transient manner. MK expression is also induced upon stimulation through the CD28 receptor alone or treatment with IL-2 or IFN-g. 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.


HIV entry into permissive cells down regulates the cell-surface expressed nucleolin (S. Nisole, B. Krust, and A.G. Hovanessian)

By using the anti-HIV pseudopeptide HB-19, we recently reported that the cell-surface expressed nucleolin is involved in the mechanism of attachment of HIV-particles to cells. HB-19 binds cells specifically and forms a stable complex with the cell-surface expressed nucleolin, resulting in the inhibition of HIV attachment. HIV particles can compete with HB-19, both in binding to the cell surface and in complex formation with surface nucleolin. These results indicate that the interaction of HIV particles with the cell-surface nucleolin is a necessary step for the attachment of HIV particles to target cells.
Following HIV attachment, here we show that the level of surface expressed nucleolin becomes reduced systematically in different types of cells infected with various HIV isolates as a consequence of viral entry process. Consistent with the implication of nucleolin in HIV entry and infection, the reduction does not occur in cells not permissive to a given virus isolate, and when entry is prevented by a neutralizing anti-CD4 mAb or by the CXCR4-specific TW70 peptide which inhibits virus to cell membrane fusion process. The decline of surface nucleolin was independent of later events in HIV infection, since it still occurred in the presence of the reverse transcriptase inhibitor AZT that exerts its effect following virus entry. Further experiments in HeLa CD4+ cells and primary macrophages infected with permissive HIV isolates demonstrated that the down regulation of surface nucleolin should be the consequence of a shift from the cell surface into the cytoplasm occurring during HIV entry. Interestingly, this shift was correlated with an increased degree of association of nucleolin with the cytoskeletal compartment. In view of these results and the characterized shuttling function of nucleolin, it is plausible to suggest that the cell-surface-expressed nucleolin acts as a carrier protein by accompanying HIV entry.


PKR and the MAP/SAP kinase signaling pathway (E. Meurs)

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system.PKR is a serine/threonine protein kinase which is strongly induced in cells after interferon treatment. PKR presents in its N terminal domain two double stranded RNA binding motifs ( DRBD). PKR acquires its kinase activity through autophosphorylation after binding to dsRNA. Once activated, PKR catalyses the phosphorylation of the a
subunit of eukaryotic protein synthesis intiation factor (eIF-2a
), which provokes inhibition of protein synthesis. Another IFN-induced, dsRNA dependent enzymatic pathway: the 2-5A synthetase/RNase L pathway, can also lead to inhibition of protein synthesis by provoking the degradation of ribosomal and messenger RNAs.
The p38 mitogen-activated protein kinases (p38 MAPKs) and the c-Jun NH2-terminal kinases (JNKs) define the stress-responsive family of the MAPK superfamily of protein kinases. These kinases are strongly activated in cells subjected to osmotic stress, UV radiation, disregulated K+ currents, RNA-damaging agents and a multitude of other stresses, like inflammatory cytokines and inhibitors of protein synthesis. Once activated, JNK phosphorylates and activates transcription factors which direct gene expression involved in cell recovery. By using murine embryo fibroblasts deficient in both PKR and RNAse L, we showed that both the PKR and RNAse L inhibition pathways are involved in JNK activation, probably through the elimination , via inhibition of protein synthesis, of a labile negative regulator (s) of the signaling to JNK acting upstream of SEK1/MKK4. They have no effect on the p38 MAPK pathway which can be activated by dsRNA through a different mechanism.


PKR and the NF-kB signaling pathway (M. Bonnet, E. Meurs)

PKR is involved in the induction of gene expression of some genes, by mediating the activation of NF-kB, an ubiquitous transcription factor. NF-kB is negatively regulated in the cytoplasm of unstimulated cells through interaction at its nuclear localization sites with the Ik
B proteins. In response to several stimuli (TNFa, IL-1b, LPS, dsRNA), IkB is phosphorylated by the IkB kinase complex (IKK), which leads to its ubiquitination and degradation by the 26S proteosome. As a consequence, NF-kB is liberated and migrates to the nucleus. The IKK complex contains two catalytic subunits IKKa and IKKb, which can form homo- or heterodimers. Another component of the multimeric IKK complex is the NF-k
B IKKb essential modulator (NEMO) which interacts with IKKb and regulates the kinase activity of IKK. IKKb is the major effector of IkB phosphorylation in response to cytokines. By using reporter assays, coimmunoprecipitationtechniques and protein/protein interactions using GST pull-down assays, we have demonstrated that PKR stimulates the NF-kB pathway without using its kinase activity, by interacting with the IKK complex.

From both studies described above, the participation of PKR in the IFN system appears to be dual: (i) as a kinase, it can regulate protein synthesis which both limits viral propagation and reinforces the induction of IFN, through JNK activation; and (ii) as a protein, it activates IKK by protein-protein interactions, leading to the NF-k
B activation and the induction of the IFN gene .


PKR and TRBP (M. Bonnet, E. Meurs)

The importance of PKR antiviral pathways is illustrated by strategies that viruses have employed to avoid the activation of PKR. These include the expression of competitive inhibitory RNAs (for instance, adenovirus VA RNAI, Epstein-Bar virus EBER RNAs) or inhibitory proteins ( for instance, vaccinia E3L and K3L, influenza NS1). PKR activation can also be blocked by protein-protein interactions with particular cellular proteins such as the TAR RNA binding protein (TRBP), p58IPK and the 60S ribosomal subunit protein L18 . TRBP has been isolated through its ability to bind HIV-1 TAR RNA. It belongs to the family of double stranded RNA binding proteins like PKR and presents two DRBD similar to those of PKR. TRBP inhibits the kinase function of PKR indirectly through sequestration of the PKR dsRNA activators and directly through heterodimerization with PKR via its DRBD. Using a reporter assay, we have confirmed that TRBP interferes with the kinase function of PKR. However, we found that TRBP cannot inhibit the PKR-mediated stimulation of NF-kB.
The physiological function of TRBP is not restricted to its ability to inhibit PKR. We have shown that TRBP stimulates in vitro the translation of RNA transcripts presenting dsRNA at their 5' terminus. By using the PKR deficient cells, we showed that the stimulation persists as TRBP can stimulate the expression from a gene reporter containing TAR at its 5' terminus.

Interaction of the HCV-encoded proteins with the antiviral action of IFN (E. Meurs)
We have used a human cell line inducible for the expression of the structural and non structural proteins of hepatitis C virus in order to study the effect of these proteins on the antiviral action of IFN. In view of recent reports indicating a role for the HCV NS5A protein as inhibitor of PKR, particular attention has been directed towards the NS5A/PKR interaction. We showed that the expression of the HCV proteins in this biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR ( by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.



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  publications

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  personnel

  Office staff Researchers Scientific trainees Other personnel
 

JEWIARZ Danièle, IP djewiarz@pasteur.fr

HOVANESSIAN Ara, CNRS/IP arahovan@pasteur.fr

KRUST Bernard, INSERM bkrust@pasteur.fr

MARIE Isabelle, IP imarie@pasteur.fr

MEURS Eliane, IP emeurs@pasteur.fr

BONNET Marion Thésard

CAILLAUD Alexandre, Thésard

LE CLECH Mikael, D.E.A.

NISOLE Sébastien, Thésard

SAID Elias, D.E.A.

ROBERT Nadine, IP

SVAB Josette, CNRS


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