Unit: Hepaciviruses

Director: Eliane MEURS

Our aim is to study the interaction of hepatitis C virus ( HCV) with its target cell and with other components of the host ( lipoproteins, immunoglobulins) and to analyse the impact of these interactions on the immune response. In the absence of efficient cellular models for HCV infection, we are setting up a strategy for the purification of human hepatocytes infected with HCV ex vivo. We are also studying the structure and the composition of viral forms circulating in the sera of HCV-infected individuals and we are characterizing the mechanism of infection of cells with this virus. By using a cellular model of HCV replicon, we are characterizing the mechanisms of inhibition of innate immune response by HCV and we are evaluating different possibilities for its restoration.

A PROCEDURE FOR THE purification of HCV-infected hepatocytes

Adrien Breiman, Catherine Ottone, Aurélie Verslype, Eliane Meurs 

(collaboration with Patrick Maurel- INSERM U128, Montpellier,Fr, Gilles Duverlie, CHU-Faculté de Médecine, Amiens, Fr ; Ceslaw Wychowski ; CNRS-FRE 2369 ; IP Lille, Fr ; Pierre Charneau, IP, Paris)

We have generated molecular tools designed to activate only in HCV-infected cells. One is a transcription factor fused to a resident protein from the endoplasmic reticulum via a cleavage motif specific of the HCV NS3/4A protease, the other is a surface protein-encoding gene placed under the control of a promoter activated by this NS3/4A-released-transcription factor. By using an HCV replicon cell line (Fig1 ), we have established the activity and specificity of the two types of constructs. Those were then transferred to pseudotyped-lentiviral vectors and introduced in primary cultures of human hepatocytes by transduction. Such hepatocytes cultures can be infected by incubation with serum from HCV patients.The ability of HCV to replicate is determined by real-time RT-PCR analysis of the negative replicative RNA strand. HCV infection was found to induce the transcription of the surface protein-encoding gene by real-time RT-PCR. Purification of HCV-infected hepatocytes will be achieved by immunomagnetic cell sorting using antibodies directed against the surface protein induced in presence of HCV. A transcriptome analysis of the purified HCV-infected hepatocytes will be carried out in order to determine which metabolic pathways are affected by HCV infection

Breiman,A., Molina,S, Pichard, L, Ottone,C., Charneau,P., Duverlie, G., Wychowski, C., Fournier,C., Maurel, P. and Meurs, E.F An HCV NS3/4A protease-dependent strategy for the identification and purification of Hepatitis C Virus-infected primary human hepatocytes. 10th International Symposium on hepatitis C virus and related viruses , December 2-6, 2003. Kyoto, Japan

STUDIES OF THE MECHANISMS OF HCV ENTRY INTO THE CELL AND OF ITS INTERACTION WITH THE " SCAVENGER " RECEPTOR TYPE B CLASS 1 (SRB1/Cla-1)

Patrick Maillard, Ursula Andréo, Agata Budkowska

(Collaboration with Therry Huby et John Chapman, U 551 Inserm Hôpital de la Pîtié, Paris)

Several candidate-receptors for HCV have been proposed on the basis of their interaction with the viral E2 envelope. However, HCV circulating in the serum of infected subjects is associated to lipoproteins, and thus could use lipoprotein receptors for its entry into target cells. We have transfected a CHO cell line, which does not naturally express any candidate HCV receptors, with a human gene encoding SR-BI, the cell receptor for lipoproteins (HDL, LDL, VLDL). This experimental system allowed us to demonstrate the capacity of HCV from the serum of infected patients to react with human SR-BI. This interaction is mediated by plasma lipoproteines associated to the virus. The function of SR-BI on human hepatic cells is under study as well as the role of the different classes of lipoproteins in the HCV entry into its target cell.

P. Maillard, T. Huby, U. Andreo, M. Moreau, J. Chapman, and A. Budkowska. The human scavenger receptor SR-BI/CLA 1 mediates binding and internalisation of natural hepatitis C virus into cells.11th International Symposium on hepatitis C virus and related viruses , October 3-7, 2004. Heidelberg, Germany.

STUDY OF THE HCV NUCLEOCAPSID AND OF ITS ROLE IN THE PATHOGENESIS OF HCV INFECTION

Patrick Maillard, Farzin Roohvand, Ursula Andréo, Agata Budkowska

(Collaborations with K .Krawczynski, Hepatitis Branch CDC Atlanta Jean -Luc Teillaud Inserm U255, Paris, Jean Pierre Lavergne, IBCP, Lyon)

The nature of the viral populations circulating in the serum of HCV infected subjects remains not well defined and infectious HCV virion has not yet been isolated and characterized. We have demonstrated that non-enveloped HCV nucleocapsids (cores) are present in the serum of infected individuals (Maillard et al. J. Virol 2001). (Fig.2). HCV nucleocapsids isolated from the serum display structure and properties similar to those of " nucleocapside-like " particles produced in insect cells infected with recombinant baculovirus. Monoclonal antibodies induced by immunisation with natural HCV nucleocapsids, allowed us to detect HCV core protein in hepatocytes of chimpazees during experimental HCV infection. (coll. avec CDC, Atlanta) (Fig.3).

The production of non-enveloped HCV nucleocapsids and their release from infected hepatocytes could be a non-conventional means to escape the host immune responses and to induce multiple immunopathological effects in the infected host. Indeed, besides their immune reactivity with specific anti-core antibodies, HCV nucleocapsids have the capacity to bind "non-immune" IgG. Via their Fcγ fragments of The " Fcγ receptor-like" site formed by the core protein mimics human "neonatal" Fcγ receptor (FcRn). Studies carried out by SPR (surface plasmon resonance) suggested that the HCV core protein can bind anti-core antibodies by " bipolar bridging " : through its paratope to the corresponding viral epitope and through its Fcγ domain to the FcγR-like motif (Fig.4). Other human viruses (such as CMV, HSV-1 EBV) encode proteins with functional properties of human FcγRs which help these viruses to avoid the effector consequences of antibody binding. The FcγR function of the viral nucleocapsid may offer HCV the possibility to escape the immune defenses mechanisms mediated by the Fcγ domain of anti-core antibodies and/or to affect functions of FcRn in the course of HCV infection. The studies carried out concern the interaction of HCV nucleocapsid and the core protein with hepatic and endothelial cells.

Maillard, J-P. Lavrgne, S. Siberil , G. Faure, F. Roohvand S. Petres , J-L. Teillaud and A. Budkowska Fcg receptor-like activity of Hepatitis C virus core protein" J. Biol. Chem. 279, 2340, 2347, 2004 .

2. A. Budkowska, P. Maillard J-P. Lavrgne, S. Siberil , G. Faure, , J-L. Teillaud Fcg receptor-like activity of Hepatitis C virus core protein" 10th International meeting on HCV and Related viruses, Kyoto, December 3-7, 2003

3.Roohvand,F., U. Andreo , M. Flamand , J.P. Lavergne, and A. Budkowska. Interaction of HCV core protein with hepatic and endothelial cells.11th International Symposium on hepatitis C virus and related viruses , October 3-7, 2004.  Heidelberg, Germany.

INTERACTION OF HCV WITH THE INNATE IMMUNE RESPONSE

Adrien Breiman, Catherine Ottone, Rachel Couderc, Romain Pierlot, Florence Bayard, Eliane Meurs

(Collaboration with John Hiscott ; Lady Davis Institute ; Montreal, Can)

Interferon-α/α (IFN-α/α) is one of the important elements of the innate immune response triggered in response to different pathogens of bacterial and/or viral origin. This response is essential for a rapid limitation of the propagation or the action of these pathogens. IFN has a direct antiviral effect by inducing several genes through the JAK/STAT signaling pathway ; it also plays an important role in the maturation and activation of dendritic cells and by linking the innate immune response to the adaptive immune response. Like other viruses (Influenza, Vaccinia, Ebola, Papillomavirus), HCV can inhibit early events in IFN induction by interfering with the phosphorylation of IRF3, a transcription factor essential for induction of IFN and of some other genes implicated in the antiviral response. HCV is therefore able to affect the host immune response which is in favor of the establishement of a chronic infection. Inhibition is mediated by its non structural protein NS3/4A. The recent identification of TBK-1 and IKK as the kinases responsable for IRF3 phosphorylation and in particular the ability of TBK-1/IKK to trigger an antiviral response prompted us to evaluate the activity of these kinases in HCV infection. We have shown that NS3/4A does not alter the intrinsic ability of TBK1 or IKK to phosphorylate IRF3 or induce IFN but acts upstream by interfering with the TRIF and RIG-I cellular signaling pathways leading to the activation of these kinases (Fig.5). We have also shown that TBK1 and IKK overexpression in an HCV replicon cell line can restore the induction of IRF3-stimulated genes, with a preferential effect for IKK. We are characterizing further the antiviral mechanism of action of IKK. Its overexpression could be used to reinforce the innate immune response in HCV infection, favour elimination of the virus from the host and prevent establishment of a chronic infection.

Breiman,A., Grandvaux,N., Lin,R., Ottone,C., Akira,S., Yoneyama, M., Fujita,.T., Hiscott, J.and Meurs,E.F Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C expression and restoration of signaling by IKKJournal of Virology ( in press)

Photos :

Figure 1

A : Schematic representation of a full length HCV replicon

The pFK-I398/neo/Core-3' plasmid containing the full length genome of HCV of genotype 1b was obtained from Pr Bartenschlager:(Pietschmann,T. et al, 2002. J Virol 76:4008-4021). The neomycine resistance gene under the control of HCV IRES at the 5' end of the construct is fused to the full length HCV genome itself placed under the control of the EMCV IRES. The HCV genome encodes for the structural proteins (Capside (C), Envelope E1 and E2, viroporine p7) and the non structural proteins (NS2, NS3, NS4A, NS4B, NS5A and the RNA-dependent RNA polymerase).

B : Expression of HCV replicon in an Huh-7 cell line

Immunofluorescence detection of some of the HCV proteins expressed from the full length replicon in the Huh-7 cell line, under selection with 100 μ g/ml of Neomycine. E2, NS3, NS4B et NS5A are revealed with antibodies kindly provided by Jean Dubuisson; CNRS-UPR2511; Lille (anti-E2), Darius Moradpour (Université de Freiburg (anti-NS3), Haralabia Boleti (Institut Pasteur Héllénique ; anti-NS4B) or with commercial antibodies (Biodesign ; anti-NS5A).

Figure 2 Analysis of HCV nucleocapsids.

Analysis of HCV nucleocapsids isolated from a patient's serum by electron microscopy. Viral particles were adsorbed to microscope grids by anti-core antibodies. Most particles are of 38-43nm in diameter. Direct staining of virus particles with 1% uranyl acetate

Figure 3.

Localisation of HCV core protein in a liver specimen from experimentally infected chimpanzee using monoclonal antibodies raised by immunisation with nonenveloped nucleocapsids purified from serum of HCV infected patient.

(a) Indirect immunofluorescence staining with Mab followed by FITC-conjugated anti-mouse IgM

(b) Analysis of liver section stained with MAb by confocal laser microscopy

Figure 4.

Schematic representation of binding of anti-core antibodies to HCV core protein by " bipolar bridging "

Binding of " non-immune " IgG to FcγR-like site via Fcγ domain

Binding of anti-core antibodies to HCV core protein by Fab and Fcγ domains at the same time by " bipolar bridging "

Figure 5. IFN induction through the TRIF and RIG-I signaling pathways

The interaction and/or entry of a virus with a cell leads to IFN induction through at least two signaling pathways : the TRIF and the RIG-I pathways. TRIF (TIR-domain-containing adapter-inducing IFN) is an adaptor protein which associates to the intracytoplasmic domain of TLR3, a member of the Toll-like receptors family, when this receptor senses te presence of extracellular double stranded RNA (dsRNA), prabably provided by the cells undergoing infection. RIG-I is an other adaptor protein which is thought to associate through its RNA helicase domain to intracytoplasmic dsRNA, such as viral RNA forms. This association would lead to a change in its conformation and provoke its interaction with other proteins via its N terminal CARD domain (CAspase Recruitment Domain). Through interaction with some still ill-defined elements, the TRIF and RIG-I pathways lead to the activation of the two protein kinases :TBK1 (TANK-binding kinase1) and IKK. Those are responsible for the phosphorylation of the trancription factor IRF3 involved in the early phase of IFN induction and in the phosphorylation of another trancription factor IRF7 (not shown) for the late phase of IFN induction. After migration to the nucleus, IRF3 activates the promoter of the IFNα gene and of some other genes (such as ISG56, ISG15, RANTES, IP-10) which can also play a role in the innate immune response.

Keywords: HCV, Interferon, NS3/4A, viral nucleocapsid, cellular receptors, SR-BI/Cla1, Fc? receptor


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