- Email: [email protected]
1157 EARLY HCVRNA DYNAMICS AND FACTORS ASSOCIATED WITH HIGH EARLY HCVRNA LEVEL DURING ACUTE HCV INFECTION
POSTERS sensitive to HCV-clinical strains infection (HCVser), support the complete replication cycle, and remain the cell-culture system that most closely mimics the in vivo situation. Nevertheless, the model is limited, mainly because few sera give measurable level of intracellular HCV RNA. These considerations prompted us to study the relationship between host cell phenotype in culture, characteristics of viral isolates and serum infectivity. Our aim was then to establish a well-defined sera collection allowing studies of HCV infection in its natural host cell and evaluation of anti-HCV compounds. Methods: From kinetic experiments, we defined the optimal conditions to infect PHH. A standard test allowed classifying ~100 sera in 3 groups, based on their infectivity toward PHH. Sera were then carefully characterized (clinical profile of patient, viral load, genotype, and cytokine/growth factor content). Results: PHH support HCVser replication for at least two weeks with high level of RNA copies per cell and de novo virus production. We found that 12% of the sera tested are highly infectious (HI; more than 5x103 HCV RNA copies/mg of total RNA), 68% are poorly infectious (PI; less than 1.3x103 copies/mg) and 20% are intermediate (Inter). Infectivity toward PHH cannot be predicted from clinical characteristics of the serum donors, HCV viral load or genotype. The HI sera have a cytokine profile that clearly distinguishes them from other groups, with low levels of the majority of the 52 analytes tested, including cytokines involved in the regulation of immune responses and inflammatory reactions. Finally, the activity of antiviral compounds was evaluated against different clinical isolates. Conclusion: We defined optimal conditions to successfully infect PHH with HCVser and showed that this highly relevant model can be useful for understanding the mechanism of HCV infection, for selection of new HCV clones able to replicate in hepatoma cell lines and to determine the potency of new antiviral drugs toward various HCV strains. 1156 THE RIBOSOMAL PROTEIN RACK1 IS A SPECIFIC HOST FACTOR REQUIRED FOR IRES-MEDIATED TRANSLATION OF HEPATITIS C VIRUS M.L. Hafirassou1,2 , K. Majzoub2,3 , S. Marzi2,4 , E. Crouchet1,2 , J.-L. Imler2,3 , T.F. Baumert1,2,5 , C. Schuster1,2 . 1 U 748 Virus-Host Interactions and Liver Disease, Inserm U 748, 2 Universit´e de Strasbourg, 3 UPR 9022 IBMC, UPR 9022 CNRS, 4 UPR 9002 IBMC, UPR 9002 CNRS, 5 Pole H´epato-Digestif, Hopitaux Universitaires de Strasbourg, Strasbourg, France E-mail: mohamed-lamine.hafi[email protected] Background: Treatment of chronic viral infection is challenged by variability of viral targets and development of resistance. Viruses depend on host factors for their life cycle, which are attractive alternative antiviral targets, provided that they are not mandatory for normal cell functions. Using a functional proteomic screen, we recently identified Receptor for Activated C Kinase 1 (RACK1) as a specific host factor required for replication of internal ribosome entry site (IRES)-containing viruses such as Drosophila C virus (DCV). Methods: Using state-of-the-art cell culture models for HCV infection, replication and translation, we investigated the functional impact of RACK1 as a host factor for HCV infection. Results: Silencing of RACK1 expression in Huh 7.5.1 cells resulted in a marked, specific and significant decrease in HCV Jc1 infection and infectious virion production. A similar effect was obtained when RACK1 expression was silenced in HCV replicating cells, demonstrating a crucial role of this host factor in HCV replication. In contrast, infection of non IRES-translated viruses like adenovirus or vesicular stomatitis virus remained unchanged in RACK1 silenced cells. In order to discriminate between the translation and the replication steps of the HCV life cycle, we established stable S470
cell lines expressing either an IRESHCV -luciferase reporter or a classical capped luciferase reporter, respectively. Silencing of RACK1 markedly and exclusively decreased IRESHCV -dependent translation, but not classical cap-mediated translation, demonstrating that RACK1 is specifically required for IRES-mediated translation of HCV. In agreement with these data, structural modeling indicates that RACK1 is located in close proximity to the HCV IRES on the 40S ribosomal subunit, in a region that is conformationally modified upon binding of the HCV IRES. Conclusions: Collectively, our results demonstrate that RACK1, a component of the ribosome, is a specific host factor for IRESdependent HCV translation. Our data conceptually advance the understanding of viral translation and reveal a novel host target for the development of antivirals addressing resistance. 1157 EARLY HCV RNA DYNAMICS AND FACTORS ASSOCIATED WITH HIGH EARLY HCV RNA LEVEL DURING ACUTE HCV INFECTION B. Hajarizadeh1 , K. Page2 , A.Y. Kim3 , B.H. McGovern4 , A.L. Cox5 , T.M. Rice2 , R. Sacks-Davis6 , J. Bruneau7 , M. Morris2 , J. Amin1 , B. Grady8 , J. Schinkel9 , L. Maher1 , M. Hellard6 , A.R. Lloyd10 , M. Prins8 , J. Grebely1 , G.J. Dore1 , on behalf of the InC3 Study Group. 1 The Kirby Institute for Infection and Immunity in Society, The University of New South Wales (UNSW), Sydney, NSW, Australia; 2 Department of Epidemiology and Biostatistics, University of California, San Francisco, CA, 3 Harvard Medical School, 4 Tufts Medical School, Boston, MA, 5 Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 6 Burnet Institute, Melbourne, VIC, Australia; 7 CRCHUM, Universit´e de Montr´eal, Montreal, QC, Canada; 8 Public Health Service of Amsterdam, 9 Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 10 School of Medical Sciences, The University of New South Wales (UNSW), Sydney, NSW, Australia E-mail: [email protected] Background and Aims: Viral dynamics during acute HCV infection can provide insights into immunopathogenesis. We aimed to assess early HCV RNA dynamics, including factors associated with high early HCV RNA level and impact on spontaneous clearance. Methods: Data were drawn from an international collaboration of nine prospective cohorts evaluating HCV infection risk and outcomes among people who inject drugs (InC3 Study). Individuals with incident HCV were identified (seroconversion within two years or symptomatic infection with seroconversion illness) and HCV RNA dynamics during acute infection evaluated. Factors associated with high early HCV RNA level (i.e. ≥6 log IU/mL, one month postinfection) were assessed and impact on spontaneous clearance evaluated using logistic regression analyses. Results: Overall, 669 participants with incident HCV were included (35% females, mean age 29 years, 49% rs12979860 IL28B CC genotype). Peak median HCV RNA levels occurred one month following infection in those with spontaneous clearance (6.0 log IU/mL, IQR=4.4–7.2) and persistence infection (5.4 log IU/mL, IQR=4.5–6.4; P = 0.091), followed by declines between months one and three of 4.2 and 0.8 log IU/mL, respectively (Figure 1A). In multivariate logistic regression among those with HCV RNA levels one month following infection (n = 189), IL28B CC genotype (vs. TT/CT, adjusted odds ratio (AOR)=3.55; 95% CI=1.67–7.56; P = 0.001) was associated with high early HCV RNA level. Age, sex and HCV genotype were not associated in adjusted analyses, but a significant interaction between IL28B genotype and sex was observed (P = 0.043). Among females, IL28B CC genotype was strongly associated with high early HCV RNA level (AOR = 6.68; 95% CI=1.73–25.73; P = 0.006), but among males, only marginally associated (AOR = 2.53; 95% CI=1.00–6.40; P = 0.050). After adjusting for age, sex, IL28B genotype and HCV genotype, high early HCV RNA
Journal of Hepatology 2013 vol. 58 | S409–S566
POSTERS level was not associated with subsequent spontaneous clearance (AOR = 1.33; 95% CI=0.48–3.69; P = 0.583). Conclusion: HCV RNA level decline was observed between one and months following infection, with continued decline among those with spontaneous clearance. IL28B genotype influenced early HCV dynamics, particularly among women, suggesting gender-specific HCV innate immune responses.
by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. Methods: To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-b induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-b mRNA expression was quantified by realtime PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic: polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-b mRNA levels and IRF-3 dimerization, induced by NDV infection. Results: Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Conclusion: A crucial region of the HCV core protein interferes with IRF-3 activation and thereby inhibits the IFN signaling cascades. Future studies involving DDX3 modification by the HCV core protein may be interesting to explore the cell growth-dysregulation mechanisms. 1159 SEQUENCING THE HEPATITIS C VIRUS: A SYSTEMATIC REVIEW B. Jacka1 , F. Lamoury1 , P. Simmonds2 , G.J. Dore1 , J. Grebely1 , T. Applegate1 . 1 Viral Hepatitis Clinical Research Program, The Kirby Institute for Infection and Immunity in Society, Darlinghurst, NSW, Australia; 2 Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK E-mail: [email protected]
Figure: Monthly medians of HCV RNA levels in acute HCV.
1158 HEPATITIS C VIRUS CORE PROTEIN BASIC AMINO ACID REGION 1 IS RESPONSIBLE FOR THE IMPAIRMENT OF IRF-3 ACTIVATION K. Inoue1,2 , M. Kohara2 . 1 Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, 2 Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan E-mail: [email protected] Aim: Hepatitis C virus (HCV) causes persistent disease in infected individuals. The innate immune system is activated immediately upon infection as the first line of host defense against invading pathogens, with type I interferon (IFN) signaling being the crucial step in the antiviral response. The IFN system is a prime target of HCV for persistent infections. IRF-3, a key transcriptional factor in the type I interferon system, is frequently impaired by HCV, in order to establish persistent infection. However, the exact mechanism
Background and Aims: Viral population sequencing has long been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of particular regions of the virus, however there has been limited standardisation of protocols. This systematic review was undertaken to map the location and frequency of population sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of population sequencing primers and amplicons to inform future research. Methods: Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (16 terms) or HCV (3 terms), plus “primer” as a general search term. Exclusion criteria included nonHCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. Results: A total of 435 studies were accepted from the 2042 identified, with 608 HCV population sequencing amplicons identified and mapped on the HCV genome. As seen in Figure 1 there is great diversity in the positioning of HCV population sequencing amplicons. The most commonly sequenced region was the Hypervariable Region-1, often utilised for studies of evolution and clustering/transmission analysis. Studies related to genotyping/classification and epidemiology favoured a defined segment of the NS5B region, likely as a result of the consensus guidelines
Journal of Hepatology 2013 vol. 58 | S409–S566
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cell lines expressing either an IRESHCV -luciferase reporter or a classical capped luciferase reporter, respectively. Silencing of RACK1 markedly and exclusively decreased IRESHCV -dependent translation, but not classical cap-mediated translation, demonstrating that RACK1 is specifically required for IRES-mediated translation of HCV. In agreement with these data, structural modeling indicates that RACK1 is located in close proximity to the HCV IRES on the 40S ribosomal subunit, in a region that is conformationally modified upon binding of the HCV IRES. Conclusions: Collectively, our results demonstrate that RACK1, a component of the ribosome, is a specific host factor for IRESdependent HCV translation. Our data conceptually advance the understanding of viral translation and reveal a novel host target for the development of antivirals addressing resistance. 1157 EARLY HCV RNA DYNAMICS AND FACTORS ASSOCIATED WITH HIGH EARLY HCV RNA LEVEL DURING ACUTE HCV INFECTION B. Hajarizadeh1 , K. Page2 , A.Y. Kim3 , B.H. McGovern4 , A.L. Cox5 , T.M. Rice2 , R. Sacks-Davis6 , J. Bruneau7 , M. Morris2 , J. Amin1 , B. Grady8 , J. Schinkel9 , L. Maher1 , M. Hellard6 , A.R. Lloyd10 , M. Prins8 , J. Grebely1 , G.J. Dore1 , on behalf of the InC3 Study Group. 1 The Kirby Institute for Infection and Immunity in Society, The University of New South Wales (UNSW), Sydney, NSW, Australia; 2 Department of Epidemiology and Biostatistics, University of California, San Francisco, CA, 3 Harvard Medical School, 4 Tufts Medical School, Boston, MA, 5 Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD, USA; 6 Burnet Institute, Melbourne, VIC, Australia; 7 CRCHUM, Universit´e de Montr´eal, Montreal, QC, Canada; 8 Public Health Service of Amsterdam, 9 Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 10 School of Medical Sciences, The University of New South Wales (UNSW), Sydney, NSW, Australia E-mail: [email protected] Background and Aims: Viral dynamics during acute HCV infection can provide insights into immunopathogenesis. We aimed to assess early HCV RNA dynamics, including factors associated with high early HCV RNA level and impact on spontaneous clearance. Methods: Data were drawn from an international collaboration of nine prospective cohorts evaluating HCV infection risk and outcomes among people who inject drugs (InC3 Study). Individuals with incident HCV were identified (seroconversion within two years or symptomatic infection with seroconversion illness) and HCV RNA dynamics during acute infection evaluated. Factors associated with high early HCV RNA level (i.e. ≥6 log IU/mL, one month postinfection) were assessed and impact on spontaneous clearance evaluated using logistic regression analyses. Results: Overall, 669 participants with incident HCV were included (35% females, mean age 29 years, 49% rs12979860 IL28B CC genotype). Peak median HCV RNA levels occurred one month following infection in those with spontaneous clearance (6.0 log IU/mL, IQR=4.4–7.2) and persistence infection (5.4 log IU/mL, IQR=4.5–6.4; P = 0.091), followed by declines between months one and three of 4.2 and 0.8 log IU/mL, respectively (Figure 1A). In multivariate logistic regression among those with HCV RNA levels one month following infection (n = 189), IL28B CC genotype (vs. TT/CT, adjusted odds ratio (AOR)=3.55; 95% CI=1.67–7.56; P = 0.001) was associated with high early HCV RNA level. Age, sex and HCV genotype were not associated in adjusted analyses, but a significant interaction between IL28B genotype and sex was observed (P = 0.043). Among females, IL28B CC genotype was strongly associated with high early HCV RNA level (AOR = 6.68; 95% CI=1.73–25.73; P = 0.006), but among males, only marginally associated (AOR = 2.53; 95% CI=1.00–6.40; P = 0.050). After adjusting for age, sex, IL28B genotype and HCV genotype, high early HCV RNA
Journal of Hepatology 2013 vol. 58 | S409–S566
POSTERS level was not associated with subsequent spontaneous clearance (AOR = 1.33; 95% CI=0.48–3.69; P = 0.583). Conclusion: HCV RNA level decline was observed between one and months following infection, with continued decline among those with spontaneous clearance. IL28B genotype influenced early HCV dynamics, particularly among women, suggesting gender-specific HCV innate immune responses.
by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. Methods: To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-b induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-b mRNA expression was quantified by realtime PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic: polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-b mRNA levels and IRF-3 dimerization, induced by NDV infection. Results: Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Conclusion: A crucial region of the HCV core protein interferes with IRF-3 activation and thereby inhibits the IFN signaling cascades. Future studies involving DDX3 modification by the HCV core protein may be interesting to explore the cell growth-dysregulation mechanisms. 1159 SEQUENCING THE HEPATITIS C VIRUS: A SYSTEMATIC REVIEW B. Jacka1 , F. Lamoury1 , P. Simmonds2 , G.J. Dore1 , J. Grebely1 , T. Applegate1 . 1 Viral Hepatitis Clinical Research Program, The Kirby Institute for Infection and Immunity in Society, Darlinghurst, NSW, Australia; 2 Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK E-mail: [email protected]
Figure: Monthly medians of HCV RNA levels in acute HCV.
1158 HEPATITIS C VIRUS CORE PROTEIN BASIC AMINO ACID REGION 1 IS RESPONSIBLE FOR THE IMPAIRMENT OF IRF-3 ACTIVATION K. Inoue1,2 , M. Kohara2 . 1 Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, 2 Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan E-mail: [email protected] Aim: Hepatitis C virus (HCV) causes persistent disease in infected individuals. The innate immune system is activated immediately upon infection as the first line of host defense against invading pathogens, with type I interferon (IFN) signaling being the crucial step in the antiviral response. The IFN system is a prime target of HCV for persistent infections. IRF-3, a key transcriptional factor in the type I interferon system, is frequently impaired by HCV, in order to establish persistent infection. However, the exact mechanism
Background and Aims: Viral population sequencing has long been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of particular regions of the virus, however there has been limited standardisation of protocols. This systematic review was undertaken to map the location and frequency of population sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of population sequencing primers and amplicons to inform future research. Methods: Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (16 terms) or HCV (3 terms), plus “primer” as a general search term. Exclusion criteria included nonHCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. Results: A total of 435 studies were accepted from the 2042 identified, with 608 HCV population sequencing amplicons identified and mapped on the HCV genome. As seen in Figure 1 there is great diversity in the positioning of HCV population sequencing amplicons. The most commonly sequenced region was the Hypervariable Region-1, often utilised for studies of evolution and clustering/transmission analysis. Studies related to genotyping/classification and epidemiology favoured a defined segment of the NS5B region, likely as a result of the consensus guidelines
Journal of Hepatology 2013 vol. 58 | S409–S566
S471