|Director : MEURS Eliane (firstname.lastname@example.org)|
The aim of this new Research Unit is to study the interaction of Hepatitis C Virus (HCV) with the cellular innate immune response, i.e; the first lines of the cellular antiviral 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. Therefore, we are setting up a strategy which will allow both the identification and the purification of HCV-infected cells. In parallel, we are studying the mechanisms of action and regulation of PKR, an interferon (IFN)-induced cellular protein kinase, which is invovled in the cellular innate immune response.
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.
Variability of the Nonstructural 5A Protein of Hepatitis C Virus type 3a Isolates and Relation to Interferon Sensitivity (in collaboration with Gilles Duverlie; laboratoire de Virologie, Université de Picardie-Jules Verne et centre hospitalo-universitaire, Hôpital-Sud, Amiens)
Interferon (as pegylated IFN) administration in combination with ribavirin remains the most successful treatment for HCV infection, with 60% of positive response. The remaining 40% can be related to the selection of virus variants that are resistant to IFN and can evade the innate immune response. Sequence analysis on a large number of isolates of HCV genotype 1b has shown a correlation between the response of HCV-infected patients to IFN and the variability of a region in the viral non structural protein NS5A. This region has been referred to as ISDR for IFN sensitivity Determining Region. Here, the entire NS5A sequence of 27 isolates of another HCV genotype (3a) was investigated in relation to the clinical response to IFN and the results confirmed the existence of a correlation between the sensitivity of the virus to IFN and the variability of the NS5A sequence (Castelain et al, 2002, J I D 185 573-83.).The mechanism of action of NS5A in the resistance of HCV to IFN, however, still remains obscure.
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-2a , 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 complex of kinases (IKK) responsible for activation of the transcription factor NF-k 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-k B, which reinforces the protection of the other cells (Bonnet et al, submitted).
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 eIF2a phosphorylation (see above § on PKR). Addition of TRBP restores translation efficiency and decreases the phosphorylation status of eIF2a , 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, J Biol Chem in press online )
Keywords: Hepatitis C Virus, cytokines, signaling, PKR, Virology
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|TALPIN Evelyne, email@example.com||Meurs Eliane,Chef laboratoire IP,firstname.lastname@example.org||BREIMAN Adrien PhD,email@example.com,
COLLINET Emilie IUT
|OTTONE Catherine, firstname.lastname@example.org