Unit: Hepaciviruses

Director: MEURS Eliane

The aim of this Research Unit is to study the interaction of Hepatitis C Virus (HCV) with its host cell and its effect on the innate immune response. Our goal is to increase the cells' ability to mount an antiviral response against HCV. At present, there is no robust cell culture system for HCV infection. HCV can infect cells of hepatocytic or hematopoietic origin, albeit with very low efficiency. Therefore, we are setting up a strategy which will allow both the identification and the purification of HCV-infected cells. HCV infection can have a negative effect on the host innate immune response, in particular through inhibition of IFN induction. We are analysing the mechanisms by which the virus inteferes with the signaling pathways involved in the induction of IFN.

Identification and purification of HCV-infected cells

(In collaboration with Patrick Maurel, INSERM U-128, & Joliette Coste, EFS, Montpellier; Gilles Duverlie; Université de Picardie-Jules Verne, Amiens ; Ceslaw Wychowski, groupe Hepatite C (CNRS, FRE 2369), Institut de Biologie de Lille & Institut Pasteur de Lille ; Pierre Charneau, Virologie Moléculaire et Vectorologie, Institut Pasteur,Paris; Haralabia Boleti, Molecular Virology Laboratoiry, Institut Pasteur Hellénique; Athènes; Grèce).

Three per cent of the world population is considered to be infected with Hepatitis C virus (HCV) with development of chronic hepatitis in 60-90% of the infected individuals, occurence of cirrhosis in 0,5 to 30% of cases and of hepatocarcinoma at a 1-3% rate/ year. HCV is an envelopped positive-strand virus which belongs to the genus hepacivirus in the Flaviviridae family. Six major HCV genotypes have been identified with several subtypes within each group and quasi-species within each subtype. Currently, there is no convenient cellular system for a robust propagation and amplification of HCV in vitro. However, even with poor efficiency (<1%), HCV is able to infect some cell cultures, including primary cultures of human hepatocytes, its natural cellular host. Therefore, we have set up a strategy in order to identify HCV-infected cells and to sort them from the bulk of hepatocytes incubated with an infectious serum. For this, we have generated constructs that activate only in presence of HCV and are designed to reveal the presence of infected cells or allow their purification.This approach will allow to (1) determine the action of HCV on cytokine signaling pathways, (2) compare the transcriptome of HCV-infected hepatocytes to that of uninfected cells, in presence or absence of interferon (IFN) treatment, one of the cytokines involved in the innate immune response and (3) determine the effect of some cellular or viral proteins on HCV replication.

Mechanism of action of PKR

PKR is an IFN-induced cellular Protein Kinase dsRNA dependent which plays an important role in the mechanisms of antiviral defense at the cellular level. PKR activates as a kinase upon binding to dsRNA. Such dsRNA structures can accumulate in cells during viral infections (i.e, as intrinseque parts of some viral genomes or as viral replicative intermediates) Once activated, PKR provokes an arrest in protein synthesis through the phosphorylation of its substrate eIF-2α , a subunit of an eukaryotic factor essential for initiation of translation. By blocking protein synthesis, PKR allows to stop or, at least, to reduce the viral dissemination through the organism. Paradoxically, PKR acts also positively on the expression of some genes and, in particular, can sustain the induction of the IFN gene. In order to do this, PKR does not require its catalytic kinase activity but directly interacts, through its N terminus, with a signaling pathway responsible for activation of the transcription factor NF-κ B . Therefore through two different mechanisms, PKR plays an important role in the antiviral action of IFN, first by blocking protein synthesis in the infected cells, and second, by sustaining the mechanisms of induction of IFN via NF-κ B, which reinforces the protection of the other cells .

The TAR RNA binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vivo independtly of its ability to inhibit the dsRNA dependent kinase PKR

(in collaboration with Catherine Vaquero, INSERM U511, Hôpital La Pitié-Salpêtrière; and Anne Gatignol; Lady Davis Institute for Medical Research; Montreal)

TRBP (HIV-1 transactivating response (TAR) RNA Binding Protein) is a cellular protein which binds dsRNA structures, like PKR. Its cDNA was initially isolated from an expression library probed with TAR RNA, a dsRNA-contaning structure present upstream of all HIV-1 (Human Immunodeficiency Virus) RNAs transcripts. Our interest in TRBP comes from its ability to inhibit the kinase activity of PKR by heterodimerisation through their respective dsRNA binding domains. Such an inhibition can easily be monitored in in vitro reticulocyte translation assays. In such assays, dsRNA-containing mRNAs are poorly translated since they activate PKRs as revealed by eIF2α phosphorylation (see above § on PKR). Addition of TRBP restores translation efficiency and decreases the phosphorylation status of eIF2α , confirming its role as PKR inhibitor. Recently, we showed that TRBP increases also the translation of dsRNA-containing mRNA independently and in addition to its ability to inhibit PKR. It may provoke a destabilization of the stable RNA structures which allows their access to the ribosomes and their efficient translation. Therefore, TRBP acts as an important cellular factor for efficient translation of dsRNA containing transcripts, both by inhibiting PKR and in a PKR-independent pathway. In the context of a viral infection, it is crucial to determine the relative effects of PKR and TRBP in order to increase the antiviral action of PKR (Dorin et al, 2003, J Biol Chem)

Keywords: Hepatitis C Virus, cytokines, signaling, PKR, Virology

Activity Reports 2003 - Institut Pasteur

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