- Email: [email protected]
45 IDENTIFICATION OF EFFECTORS OF THE INTERFERON-INDUCED ANTIVIRAL RESPONSE AGAINST HEPATITIS C VIRUS
ORAL PRESENTATIONS 43 PACKAGING OF HEPATITIS E VIRUS (HEV) GENOMIC RNA IN TRANS-COMPLEMENTED CAPSID PROTEIN AND PRODUCTION OF INFECTIOUS VIRIONS M.K. Parvez1,2 , S.U. Emerson1 , R.H. Purcell1 . 1 Molecular Hepatitis Section, Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, MD, USA; 2 Department of Pharmacognosy, King Saud University College Pharmacy, Riyadh, Saudi Arabia E-mail: [email protected] Background and Aim: In the absence of a robust in vitro as well as in vivo model, a recently developed HEV (sar55) genomic replicon system (pSK-GFP) has allowed to study and elucidate some important aspects of its molecular biology. However, partial swapping of viral capsid (ORF2)/ non-structural (ORF3) proteins by the GFP marker abolishes their expressions and hence packaging of the genomic RNA. We therefore, intended to develop an in vitro HEV RNA packaging system that could produce infectious viral particles. Methods: A recombinant baculovirus (vBac-ORF2, titre-10E10 pfu/ ml)) was constructed (pSK-E2; HEVsar55-ORF3/ORF2; nt. 5130– 7204). Expression of HEV capsid protein (pBac-ORF2) was tested in vitro using TNT Coupled Reticulocyte Lysate System (Western analysis) as well as in vBac-ORF2-transduced S10-3 cells (immunefluorescence microscopy). pSK-GFP was In vitro transcribed and HEV RNA was transfected into S10-3 cells and viral replication was determined by GFP expression (fluorescence microscopy and flow cytometry). Transduction of vBac-ORF2 was done post-transfection followed by S10-3 lysate preparation on day6. The intracellular virions in the lysates were used to infect naive HepG2 cells and production of infective particles were determined. Results: Western blot analysis showed expression of expected full length ORF2 (~72 kDa) as well as a processed form of ~55 kDa in S10-3 cells. The FACS analysis of HEV RNA replication showed ~16% of GFP positivity in transfected S10-3 cells at day6. HepG2 cells when analyzed by FACS, showed ~2% positivity for GFP at day6 post-infection, indicating production of 1/8th (12.5%) of stable infectious particles. In accordance with our fluorescence microscope observations, naive cells infected with lysates from moc-transfected, RNA-transfected or Bac-ORF2-transduced cells scored negative for GFP. We however, did not detect any infectious particles in the culture media. Conclusions: 1. Our results, for the first time, show that the HEV capsid over-expressed in mammalian system is biologically active and efficently encapsidtes HEV RNA to produce stable infectious virions. 2. This valuable system could be further used to study many aspects of HEV biology. 44 INTERFERON-b AND -l SIGNALING IS NOT AFFECTED BY INTERFERON-INDUCED REFRACTORINESS TO INTERFERON-a IN VIVO Z. Makowska1 , F.H. Duong1 , G. Trincucci1 , M.H. Heim1,2 . 1 Department of Biomedicine, University Basel, 2 Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland E-mail: [email protected] Background and Aims: Therapy of chronic hepatitis C (CHC) with pegylated interferon a (pegIFN-a) and ribavirin achieves sustained virological responses in approximately half of patients. Nonresponse to treatment is associated with constitutively increased expression of interferon stimulated genes in the liver already before therapy. This activation of the endogenous IFN system could prevent cells from responding to therapeutically injected (peg)IFN-a, because prolonged stimulation of cells with IFN-a induces desensitization of the IFN signal transduction pathway. Whether all types of IFNs induce refractoriness in the liver is presently unknown. S20
Methods: We initially investigated the responses of Huh7 cells to repeated treatments with different doses of human IFN-a, IFN-b and IFN-l. Based on these findings, mice were treated with multiple injections and different combinations of IFN-a, IFN-b, IFN-g and IFN-l. Signal transduction through the Jak-STAT pathway in response to these treatment regimens was analyzed with Western blots, electrophoretic mobility shift assays (EMSAs) and quantitative RT-PCR of interferon target gene transcripts in mouse liver and gut samples. Finally, the response to IFN-a and IFN-l was assessed in ex vivo treated human liver biopsies. Results: Pretreatment of mice with IFN-a, IFN-b and IFN-l induced a strong expression of the negative regulator USP18 in the liver and gut. As a consequence, IFN-a signaling was significantly reduced when mice where re-injected 16 hours after the first injection. Surprisingly, both IFN-b and IFN-l could activate the Jak-STAT pathway and the expression of ISGs despite high levels of USP18. IFN-l treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN-a induced STAT1 activation was transient. Conclusions: Contrary to IFN-a, IFN-b and IFN-l signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN-b and IFN-l could be an important advantage for the treatment of pegIFN-a non-responder patients with a pre-activated endogenous IFN system. 45 IDENTIFICATION OF EFFECTORS OF THE INTERFERON-INDUCED ANTIVIRAL RESPONSE AGAINST HEPATITIS C VIRUS P. Metz1 , E. Dazert2 , J. Mazur3 , M. Frese4 , L. Kaderali3 , V. Lohmann1 , R. Bartenschlager1 . 1 Molecular Virology, University of Heidelberg, Heidelberg, Germany; 2 Biozentrum, Growth & Development, University of Basel, Basel, Switzerland; 3 Viroquant Research Group Modeling, University of Heidelberg, Heidelberg, Germany; 4 Faculty of Appied Science, University of Canberra, Canberra, ACT, Australia E-mail: [email protected] Persistent infection with the hepatitis C virus (HCV) may lead to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Current therapy relies on the antiviral activity of interferon-alpha (IFN-a) that is given alone or in combination with ribavirin. However, treatment outcomes for the most prevalent genotypes are still not optimal, and side effects limit therapy success. Moreover, antiviral T cell responses targeting HCV appear to be mediated primarily by IFN-g, but the effectors responsible for replication inhibition are not known. IFNs induce the expression of a multitude of IFN-stimulated genes (ISGs), but for the majority, little is known about their anti-HCV potential and mode-of-action. Here we report a novel RNAi-based screening assay designed to identify ISGs contributing to the inhibition of HCV replication. The assay is based on the restoration of viral replication in the presence of IFNs by knockdown of ISGs. In this way we identified 6 ISGs with previously unreported antiviral activity. One of them was specific for IFN-a, one for IFN-g and 4 were induced by either cytokine, demonstrating a substantial overlap of the respective signalling and effector pathways. Combinatorial knockdowns of these ISGs after IFN treatment suggest additive effects. In conclusion, our study identifies several ISGs contributing to the block of HCV replication by type 1 and type 2 IFN and they suggest that inhibition is conferred by the combined action of several ISGs.
Journal of Hepatology 2011 vol. 54 | S1–S24
Methods: We initially investigated the responses of Huh7 cells to repeated treatments with different doses of human IFN-a, IFN-b and IFN-l. Based on these findings, mice were treated with multiple injections and different combinations of IFN-a, IFN-b, IFN-g and IFN-l. Signal transduction through the Jak-STAT pathway in response to these treatment regimens was analyzed with Western blots, electrophoretic mobility shift assays (EMSAs) and quantitative RT-PCR of interferon target gene transcripts in mouse liver and gut samples. Finally, the response to IFN-a and IFN-l was assessed in ex vivo treated human liver biopsies. Results: Pretreatment of mice with IFN-a, IFN-b and IFN-l induced a strong expression of the negative regulator USP18 in the liver and gut. As a consequence, IFN-a signaling was significantly reduced when mice where re-injected 16 hours after the first injection. Surprisingly, both IFN-b and IFN-l could activate the Jak-STAT pathway and the expression of ISGs despite high levels of USP18. IFN-l treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN-a induced STAT1 activation was transient. Conclusions: Contrary to IFN-a, IFN-b and IFN-l signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN-b and IFN-l could be an important advantage for the treatment of pegIFN-a non-responder patients with a pre-activated endogenous IFN system. 45 IDENTIFICATION OF EFFECTORS OF THE INTERFERON-INDUCED ANTIVIRAL RESPONSE AGAINST HEPATITIS C VIRUS P. Metz1 , E. Dazert2 , J. Mazur3 , M. Frese4 , L. Kaderali3 , V. Lohmann1 , R. Bartenschlager1 . 1 Molecular Virology, University of Heidelberg, Heidelberg, Germany; 2 Biozentrum, Growth & Development, University of Basel, Basel, Switzerland; 3 Viroquant Research Group Modeling, University of Heidelberg, Heidelberg, Germany; 4 Faculty of Appied Science, University of Canberra, Canberra, ACT, Australia E-mail: [email protected] Persistent infection with the hepatitis C virus (HCV) may lead to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Current therapy relies on the antiviral activity of interferon-alpha (IFN-a) that is given alone or in combination with ribavirin. However, treatment outcomes for the most prevalent genotypes are still not optimal, and side effects limit therapy success. Moreover, antiviral T cell responses targeting HCV appear to be mediated primarily by IFN-g, but the effectors responsible for replication inhibition are not known. IFNs induce the expression of a multitude of IFN-stimulated genes (ISGs), but for the majority, little is known about their anti-HCV potential and mode-of-action. Here we report a novel RNAi-based screening assay designed to identify ISGs contributing to the inhibition of HCV replication. The assay is based on the restoration of viral replication in the presence of IFNs by knockdown of ISGs. In this way we identified 6 ISGs with previously unreported antiviral activity. One of them was specific for IFN-a, one for IFN-g and 4 were induced by either cytokine, demonstrating a substantial overlap of the respective signalling and effector pathways. Combinatorial knockdowns of these ISGs after IFN treatment suggest additive effects. In conclusion, our study identifies several ISGs contributing to the block of HCV replication by type 1 and type 2 IFN and they suggest that inhibition is conferred by the combined action of several ISGs.
Journal of Hepatology 2011 vol. 54 | S1–S24