Director: Eliane MEURS
Our aim is to investigate the interactions of hepatitis C virus (HCV) and its structural components (such as viral nucleocapsid) with its target cell and with host molecules (lipoproteins, immunoglobulins) and the role of these interactions in the physiopathology of infection. We analyse the structure and composition of viral forms circulating in the serum of HCV-infected individuals and mechanisms of cell infection. In particular, we investigate the role of lipoproteins in HCV cellular entry and in the recognition of hepatic receptors by this virus.
We investigate equally HCV cellular permissivity. In order to to this, we have set up a strategy for the identification and the purification of human hepatocytes infected with HCV ex vivo, which is based on the activity of the HCV NS3/4A protease.
HCV interferes with IFN induction and action, a major mechanism of the host immune response. Indeed, a number of HCV-infected patients do not respond to the anti-viral treatment (pegylated IFN + ribavirin). We study therefore mechanisms by which HCV interferes with the cellular signalling pathways involved in interferon induction using a model of HCV replicon and a cell culture system infected with recombinant HCV.
MECHANISMS OF HCV CELLULAR ENTRY, ITS Interaction WITH " scavenger " RECEPTOR (SRB1/Cla-1) AND role OF lipoproteines
Patrick Maillard, Ursula Andréo, Olga Kalinina, Agata Budkowska (Collaborations : Therry Huby et John Chapman, U 551 Inserm, Hôpital de la Pîtié, Paris ; Patrick Marcellin et Michèle Martinot, Hôpital Beaujon, Paris ; Jean-Michel Pawlotsky , Hôpital Henri Mondor, Créteil)
Several candidate-receptors for HCV have been proposed on the basis of their reactivity with the E2 envelope glycoprotein. However their role in the cell infection with natural HCV circulating in the serum of HCV infected patients remains to be determined. Human " scavenger " receptor SR-BI/Cla1 is a cellular receptor for HDL, LDL VLDL which also facilitates cholesterol efflux to these lipoproteins. HCV circulating in the serum of infected individuals is associated with different classes of lipoproteins (LDL, VLDL, HDL), and therefore it could use lipoprotein receptors for cell entry. We demonstrated that human " scavenger " receptor SR-BI/Cla1, expressed on CHO cell line transfected with a gene encoding human receptor, and on human hepatoma cells has a capacity to bind and internalise natural HCV from serum of HCV-infected patients. This interaction is not mediated by E2 envelope glycoprotein - since it is not inhibited by anti-envelope antibodies- but is mediated by lipoproteins (mainly VLDL) associated to the virus. Indeed, physiological SR-BI/Cla1 ligands such as VLDL,LDL, and anti β- lipoprotein antibodies inhibit HCV cellular entry in vitro. Our results suggest the existence of the alternative way of cell infection by HCV which involves apoB lipoproteins (VLDL et LDL) associated to the virus and lipoprotein receptors on the hepatocyte: SR-BI/Cla1 et LDL-R. This way of infection would permit the virus cell infection in the presence of neutralising antibodies and could represent the mechanism of subversion of the host immune response (1) (Figure 1) .
Studies currently carried out aim to determine the mechanism of cell infection by HCV and the role of lipoproteins and other molecules involved in lipoprotein metabolism in cellular entry of the virus. (2, 3).
1. P. Maillard , T. Huby U. Andreo , M. Moreau J. Chapman , and Agata Budkowska ; Interaction of natural hepatitis C virus with the human scavenger receptor SR-BI/Cla1 is mediated by apo-B-containing lipoproteins FASEB J, Feb, 2006
2. P.André, G. Perlemutter, A. Budkowska, C. Bréchot and V. Lotteau. Hepatitis C virus and lipid metabolism. Seminars in Liver disease 25 (1), 95-1004, 2005.
3. U. Andréo , P. Maillard, M. Martinot, P. Marcellin, and A. Budkowska Manuscript en preparation.
STUDY OF THE HCV NUCLEOCAPSID AND OF ITS ROLE IN THE PATHOGENESIS OF HCV INFECTION
Patrick Maillard, Farzin Roohvand, Ursula Andréo, Agata Budkowska (Collaborations : K .Krawczynski, Hepatitis Branch CDC Atlanta Jean Luc Teillaud Inserm U255, Paris, Jean Pierre Lavergne, IBCP, Lyon, Grazyna Faure, IP Paris, M. Flamand IP Paris, Jacques d'Alayer IP Paris)
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 from serum and characterized. We have demonstrated that non-enveloped HCV nucleocapsids (cores) are present in the serum of infected individuals (4). (Figure.2). Monoclonal antibodies induced by immunisation with these viral particles, allowed us to detect HCV core protein in hepatocytes of chimpazees during experimental HCV infection. (coll. avec CDC, Atlanta) (Figure.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. 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 (Figure.4). Other human viruses (such as CMV, HSV-1 EBV) encode proteins with functional properties of human FcγRs which might 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.
Current studies concern the interaction of the core protein with hepatic and endothelial cells and in particular with cytoskeleton components ( 6).
4. P.Maillard, K. Krawczynski, J. Nitkiewicz, Ch..Bronnert, M. Sidorkiewicz, P.Gounon, J. Dubuisson, G.Faure, R.Crainic and A.Budkowska, Non-enveloped nucleocapsids in serum of HCV infected patients. J. Virol. 75, 8240-8250, 2001
5. P.Maillard, J-P. Lavergne, S. Siberil , G. Faure, F. Roohvand S. Petres , J-L. Teillaud and A. Budkowska Fcγ receptor-like activity of Hepatitis C virus core protein" J. Biol. Chem. 279, 2340, 2347, 2004 .
6. F. Roohvand, JP Lavergne, U Andréo, J. D'Alayer, and A. Budkowska. manucscript in preparation..
IMPACT OF HCV VARIABILITY ON DIAGNOSTICS OF HCV INFECTION
Patrick Maillard, Olga Kalinina , Agata Budkowska. PTR project #126 coordinated by Penelope. Mavromara . Collaboration with IP Athens, IP St. Petersburg, IP Cambodge, IP Vietnam, IP Cameroon, Institut Cantacuzène ( Rumania).
In collaboration with other Institutes from the RIIP (Réseau International des Instituts Pasteur), we investigate the impact of HCV genotypes, other than genotype 1 on the detection of anti-HCV antibodies in patients' sera. HCV genotypes such as genotype 4 and 6 are highly prevalent in South East Asia and Central Africa and Eastern Europe. The main goals of this project are : (1) to evaluate the impact of genetic variability of the virus on the performance of diagnostics of infection in this parts of world and (2) to produce new reagents and development of new methods to improve existing diagnostic assays.
A PROCEDURE FOR THE IDENTIFICATION AND PURIFICATION OF HCV-INFECTED HEPATOCYTES.
Adrien Breiman, Damien Vitour, Myriam Vilasco, Eliane Meurs (collaboration avec Patrick Maurel- INSERM U128, Montpellier, Gilles Duverlie, CHU-Faculté de Médecine, Amiens; Czeslaw Wychowski ; CNRS-FRE 2369 ; IP Lille, ; Pierre Charneau, IP, Paris )
Our goal is to identify the HCV-infected cells in order to determine the cellular characteristics of permissivity to infection. In order to do this, a chimeric form of the Gal4VP16 transcription factor was engineered to activate only in presence of the HCV NS3/4A protease and induce different reporter genes (CAT, GFP and the cell surface H-2 Kκ marker) through the (Gal4)5-E1b promoter. (Figure 5). For this, the NS5A/5B transcleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK and we demonstrated that it could be specifically cleaved by NS3/4A. Accordingly, transient transfection in the Tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a, revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in the UHCV-11 cells and then in the Huh-7 cells expressing an HCV replicon of genotype 1b. In addition, the GFP reporter gene allowed rapid fluorescent monitoring of HCV expression in the replicon cells. HCV infects human hepatocytes in primary cultures with poor efficiency. By using the H-2 Kκ cell surface marker as gene reporter and by introducing the different constructs (chimera and reporter genes) by transduction using VSV pseudotyped lentiviral vectors, it is now possible to purify HCV-infected hepatocytes by immunomagnetic cell sorting. It is however difficult to obtain hepatocytes on a regular basis (from surgical resections). However, the cell sorting could be validated thanks to the possibility to infect cell lines of hepatic origin with HCv of genotype 2a that can be generated in cell culture (HCV JFH1). The chimera activation system could be demonstrated using first the CAT reporter assay and then the H-2 Kκ reporter gene which allowed a selective sorting of the HCV-infected cells (Figure 6). We are now performing the analysis of the cellular characteristics which are favourable for HCV infection.
A. Breiman, D. Vitour, M.Vilasco, D. Delgrange, C. Ottone, S. Molina, L. Pichard-Garcia, C. Fournier, P. Charneau, G. Duverlie, C. Wychowski, P. Maurel and E. F. Meurs An HCV NS3/4A protease-dependent strategy for the identification of Hepatitis C Virus-infected cells submitted
INTERACTION OF HCV WITH THE INNATE IMMUNE RESPONSE
Adrien Breiman, Damien Vitour, Myriam Vilasco, Stéphanie Dabo, Eliane Meurs (Collaboration avec John Hiscott et Rongtuan Lin ; Lady Davis Institute ; Montréal, Can, Plateforme " Puces à ADN ", Institut Pasteur, Pierre Eid, CNRS, UPR 9045, Villejuif; Jesus Prieto, CIMA, Pampelone, Espagne)
A direct antiviral role for IKKe in HCV infection
We have recently shown that the HCV NS3/4A protease can interfere with two dsRNA-mediated IFN inducing pathways, the TRIF and the RIG-I pathways .Both lead to the activation of the NF-κB and IRF3 transcription factors via the IKKαβ and TBK1/IKKe protein kinases and it is now known that the HCV NS3/4A protease cleaves TRIF and dissociates RIG-I form the downstream kinases by cleaving the mitochondrial-linked adapter Cardif (Figure 7). By overexpressing IKKe in an HCV replicon cellular model, we have shown that IKKe can inhibit HCV expression, in accord with the function of this kinase downstream of the NS3/4A blockade . In those conditions, we have established by microarray, the IKKe -induced transcriptome in the replicon cells and we have shown a strong induction of genes known to be induced also by IFN and to have an antiviral role. Moreover, IKKe still inhibits HCV expression in presence of antibodies directed against the IFN receptor, which suggests its rapid participation to the cellular innate immune response, en addition to its role in IFN induction. In order to know more on the physiological role of IKKe in the cellular response to HCV infection, we have analysed its expression level in liver biopsies from HCV-infected patients. Analysis of the RIG-I levels as well as those of other genes involved in the RNA helicase-mediated induction pathway (MDA5, LGP2, Cardif, TBK1), IFNβ and two IKKe -induced genes, were also included in this study. The results show that HCV infection strongly inhibits the IKKe and RIG-I expression levels, as compared to other genes involved in the IFNβ induction pathway. These data argue in favour of an important role for IKKe and RIG-I in the innate immune response. Their inhibition upon HCV infection can explain, at least in part, the HCV persistence in the host (4).
Analysis of the interaction of IKKe with the mitochondrial adapter Cardif
In accord with the importance of IKKe in the innate immune response of the host, this knase has been recently shown to be specifically localized with the RIG-I-recruited adapter Cardif, whereas TBK1 is not. . At present, we are characterizing the Cardif motfis which are necessary for its interaction with IKKe . We are also establishing which are the cellular partners involved in the IKKe / Cardif interaction by library screening in a yeast two-hybrid assay. .
(3) Lin, R., J. Lacoste, P. Nakhaei, Q. Sun, L. Yang, S. Paz, P. Wilkinson, I. Julkunen, D. Vitour, E. F. M e u r s, and J. Hi s c o t t. Dissociation of a MAVS/IPS/VISA/Cardif-IKke molecular complex from the mitochondrial outer membrane by hepatitis virus C NS3/4A proteolytic cleavage. J Virol. in press
(5) Vitour, D., S. Dabo, M. VIlasco, and E. F. Meurs. in preparation
Legends to Figures
Figure 1. HCV associated with VLDL (1) binds to SR-BI/Cla1 via VLDL (2). Anti-SR-BI antibodies (3), VLDL (4) and anti-b lipoprotein antibodies (5) inhibit the interaction of HCV with SR-BI/Cla1, whereas anti-E2 and anti-HVR1 antibodies do not (6) (From FASEB J, February 2006) .
Figure 2 Microscopic electronic analysis of HCV nucleocapsids isolated from patient sera. The viral particles were adsorbed on the grid through anti-core antibodies. The nucleocapsids were in majority 38 to 43nm in diameter. Direct coloration with 1% Uranyl acetate (J Virol. 75 8240-8250, 2001).
Figure 3. Localization of the HCV core protein in liver from chimpanzee during HCV infection. Indirect immunofluorescence using a monoclonal antibody produced by immunization with non-envelopped nucleocapsids purified from a patient'serum.
(a) Indirect coloration with anti-core Mab anti-capside and revelation with FITC-conjugated murine anti-IgMs.
(b) Confocal microscopy analysis of hepatic tissue labelled with anti-core Mab anti-capside (J Virol. 75 8240-8250, 2001).
Figure 4. Schematic représentation of the binding of an anti-core antibody to the HCV core protein by " bipolar bridging "
Attachment of "non-immune " IgGs on the " Fcγ R-like " site via the Fcγ domain ; Attachment of the anti-core antibodies on the core protein through the Fab and Fcγ domains at the same time by "bipolar brigding ".
Figure 5 - Strategy for the identification and purification of HCV-infected cells.
The Gal4VP16 transfection factor is fused to an endoplasmic reticulum resident protein through the central part of the NS5A/5B motif sensitive to the protease action of NS3/4A (Chimera ).Different reporter genes (CAT, GFP, or H2Kk) have been placed under the control of the Gal4-VP16 inducible promoter (Gal4)5-E1b. Upon expression of NS3/4A, the Gal4VP16 moiety of the chimera is cleaved and migrates to the nucleus where it can induce the expression of reporter genes. Expression of CAT is determined by ELISA, that of GFP by fluorescence and immunoblot. Expression of H2Kk at the cell surface was used for immunomagnetic cell sorting of HCV-expressing cells.
Figure 6 : Purification of HCV-infected cells
Huh7 cells were infected with a preparation of JFH1 HCV and transduced 24h after the onset of infection with VSV-pseudotyped lentiviral vectors HR'TRIP expressing the selection markers,including the the murine cell surface marker H2Kk.After three days of culture, the H2K κ positive cells were sorted by AUTOMACS. A control of HCV infection was performed in parallel by the titration of viral foci using serum from an HCV-infected patient.
Figure 7 : Interference of HCV with the IFN inducing pathways
During a viral infection, the dsRNA-mediated induction of IFN is triggered either through interaction with the extracytoplasmic domain of the Toll-like receptor 3 (TLR3) or, after a viral intrusion, through interaction with specific cellular RNA helicases such as RIG-I. In both cases, there is activation of the MAPK, IKKαβγ and TBK1/IKKe protein kinases, which provokes activation of the transcription factors AP-1, NF-κ B et IRF3, respectively, and IFN induction . In regards with the TLR3 activation pathway, there is recruitment of the TRIF adapter through the intracytoplasmic domain of TLR3. In regards with the RIG-I activation pathway, the binding of the RIG-I RNA helicase domain to dsRNA provokes a change in its conformation which allows its N terminal CARD domain to bind the recently identified IPS-1/MAVS/VISA/Cardif protein which is bound to the mitochondrial membrane . This adapter is responsible for the recruitment of the downstream kinases which phosphorylate AP-1, NF-κ B etIRF3. The genome of the Hepatitis C virus presents structured dsRNA regions, such as its 5'NTR and 3'NTR which can trigger IFNβ induction soon after introduction in the cellular host . However, HCV can interfere with IFN induction through the action of its NS3/4A protease which can cleave the mitochondrial MasterCARD adapter . In addition, the NS3/4A protease can cleave the TRIF adapter , thus emphasizing the importance of these two IFN signalling pathways and the necessity for the virus to inhibit these pathways in order to favour its own propagation.
Keywords: VHC, Interferon, NS3/4A, RIG-I, Cardif, IKKε, viral nucleocapsid, HCV cellular receptors récepteurs, SR-BI/Cla1, Fcγ receptor