We investigate the interactions of Hepatitis C virus (HCV) and Hepatitis B virus (HBV) with their cellular host in view of developing new approaches to inhibit their replication, propagation and maintenance, as chronic infection with both viruses represent major risk factors for the development of hepatocellular carcinoma. Ongoing projects include the characterization of the interactions of HCV with components of the Interferon-inducing pathway, of the mechanisms involved in HCV cell entry and its intracellular transport, and the characterization of cellular effectors involved in HBV replication and pathogenesis.

HCV infection and the control of the Interferon (IFN) inducing pathway (E.Meurs)

Cells can sense unusual DNA, ssRNA or dsRNA structures, such as those produced during viral infections, through specific Pathogen Recognition Receptors (PRRs). This triggers induction of IFN and of pro-inflammatory cytokines, activating the innate immune response of the host. One of the signalling pathways leading to IFN induction involvesthe cytosolic RNA helicase RIG-I as PRR, its interaction with the mitochondria-bound adapter MAVS and the recruitement of the NF-κB-and IRF3/7-activating kinases: IKKβand the TBK1/IKKεkinases. We characterized the mode of interaction of IKKεwith MAVS and showed the importance of IKKεin regulating the ability of MAVS to activate the NF-κB pathway(Mol.Cell.Biol, 2009). Through a yeast two-hybrid search, we identified the cell cycle-related kinase PLK1 as a novel regulator of MAVS and IKKε, establishing a novel link between innate immunity and cell cycle (J.Biol.Chem, 2009).HCV is a poor IFN inducer, despite recognition of its RNA by RIG-I. This is due in part through cleavage of MAVS by the HCV NS3/4A protease. In addition, we showed that HCV controls IFN production through activation of the eIF2αkinase PKR (PlosOne, 2010) and through the di-ubiquitine like protein ISG15 (MS in preparation).These results provide new insights in the interaction of HCV with the innate immune pathway and point out to PKR and ISG15 as novel targets to restore IFN induction during HCV infection.

Role of lipoproteins, lipoprotein receptors and the microtubules in HCV infection (A. Budkowska).

HCV represents unique model of virus cell- interaction due to the central role of lipoproteins in the viral life cycle (J.gen Virol 2009). We showed that due to its association with VLDL, HCV is a target for the lipoprotein lipase (LPL)- an enzyme mediating uptake of lipoproteins by the liver. LPL revealed to be a potent inhibitor of cell infection with HCV produced in vitro (Huh7.5 cells) and in chimeric mice with transplanted human hepatocytes (MS in preparation). We showed that HCV requires microtubule network for the initiation of a productive infection. HCV core protein directly interacts with tubulin increasing microtubules polymerisation. HCV can thus exploit the microtubule polymerization mechanisms, to promote virus entry and transport (J.Biol. Chem. 2009). Our studies suggest that virus-associated lipoproteins and microtubules could be considered as attractive targets for therapeutic interventions against HCV infection. In collaboration with several Pasteur Institutes from the Réseau we proposed NS4B proteinbased assays to improve detection of particular HCV genotypes (Clin.l Microbiol 2010). We also showed that synonymous mutations in the core gene could be correlated with an unusual serological profile in HCV infected patients. These mutations induce changes in the secondary structure of the core region of HCV RNA, which promote production of the unusual nucleocapsid protein and overexpression of a novel protein encoded by the alternative open reading frame of HCV genome (PloSOne 2011).

Cellular partners of the viral protein HBx in viral replication and oncogenesis(Christine Neuveut)

A fundamental question in HBV field is to understand the complex interplays between HBV and the host that determine the outcome of infection and cancer development. The HBV-encoded HBx oncoprotein is a multi-functional regulator of transcription and signal transduction that is essential for HBV replication. We have demonstrated functional cooperation between HBx and cellular chromatin modifying complexes such as the histone acetyltransferase p300/CBP and a complexe containing the deacetylase HDAC1 and PP1, reinforcing the notion that HBx is involved in the epigenetic regulation of transcription, impacting both on virus replication and on the cellular environment.We are currently studying the architecture of theses complexes and investigating their role in the transcriptional activity of HBx and in the epigenetic control of cccDNA as well as of cellular genes upon HBV infection. A new study has been launched to examine HBx interaction with the DDB1 subunit of the Cul4A ubiquitin E3 ligase complex. This complex might be subverted by HBx to benefit viral replication. Through crystal structure and proteomic analysis, we are searching for cellular substrates selectively targeted by the HBx/DDB1 complex.

Keywords: Hepatitis C virus, lipoproteins, microtubules, nucleocapsid, alternative ORF protein , PLK1, MAVS, Interferon, PKR, ISG15, HBX, transcription, epigenetic regulation, DDB1, PP1

1. Andreo, U., P. Maillard, O. Kalinina, M. Walic, E.F. Meurs, M. Martinot, P. Marcellin, P., and A. Budkowska. 2007. Lipoprotein lipase mediates hepatitis C virus (HCV) cell entry and inhibits HCV infection. Cellular Microbiology 9, 2445-2456. (PMID: 17517063).

2. F. Roohvand, P.Maillard, JP Lavergne, S. Boulant, M. Walic, U. Andréo, L.Goueslain, F. Helle, A.Mallet, J. McLauchlan and A. Budkowska. 2009. Initiation of hepatitis C virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein. J Biol Chem 284(20):13778-91. (PMID: 19269968)

3. Paz, S., M. Vilasco, M. Arguello, S. Q, J. Lacoste, T. Nguyen, T. Zhao, S. Zaari, A. Bibeau-Poirier, M. J. Servant, R. Lin, E. F. Meurs, and J. Hiscott. (2009). Ubiquitin-regulated recruitment of IKKε to MAVS interferon signaling adapter. Mol Biol Cell. 29 : 3401-12. (PMID: 19380491)

4. Vitour, D., S. Dabo, M. Ahmadi Pour, M. Vilasco, P. O. Vidalain, Y. Jacob, M. Mezel-Lemoine, S. Paz, M. Arguello, R. Lin, F. Tangy, J. Hiscott, and E. F. Meurs. 2009. Polo-like kinase 1 (PLK1) regulates interferon (IFN) induction by MAVS. J Biol Chem 284:21797-809. (PMID: 19546225)

5. Arnaud, N., Dabo, S., Maillard, P., Budkowska, A., Kalliampakou, K. I., Mavromara, P., Garcin, D., Hugon, J., Gatignol, A., Akazawa, D., Wakita, T., Meurs, E.F. (2010). Hepatitis C Virus controls Interferon production through PKR activation. Plos One, May 11;5(5):e10575. (PMID: 20485506)