- Email: [email protected]
Hepatocyte single cell sequencing to identify transcript modulation by hepatitis C virus
POSTER PRESENTATIONS Recerca-Hospital Universitario Vall d’Hebron (VHIR-HUVH), Barcelona; 6 Centro de Biología Molecular “Severo Ochoa” (CBMSO), Consejo Superior de Investigaciones Científicas (CSIC-UAM); 7Instituto de Genética Médica y Molecular (INGEMM), Madrid, Spain E-mail: [email protected] Background and Aims: Previous studies have shown that hepatitis C virus (HCV) induces epigenetic changes through interaction with Aurora B kinase (AURKB). These epigenetic effects correlate with the inhibition of the inflammatory pathway genes [1]. Our aim was to analyze the genetic variation of AURKB and to determine its potential clinical value as prognostic marker of liver fibrosis progression (LFP). Methods: We included 348 patients with chronic hepatitis C (CHC): 26.7% F0-F1 (93/348); 33.6% F2 (117/348), 15.2% F3 (53/348) and 24.4% F4 (85/348). The clinical features were: gender (M/F): 182/166; age (mean ± SD): 58.55 ± 11.9; HCV genotype: 1a: 62 (17.8%), 1b: 154 (44.3%), 2: 1 (0.3%), 3: 27 (7.8%), 4: 18 (5.2%), 5: 1 (0.3%), and undetermined: 85 (24.4%). We analyzed by bulk sequencing the frequency of SNPs in the rs2241909 and rs1059476 positions of AURKB gene. Allelic distribution was compared between noncirrhotic (F0–F3) vs cirrhotic (F4) patients and also between low (F0–F2) vs high grade (F3–F4) of fibrosis. Results: Significant differences were observed in the distribution of alelles C/T in both rs2241909 and rs1059476 positions. The rate of heterozygosis in rs2241909 was significant lower in non-cirrhotic vs cirrhotic patients [110/257 (42.8%) vs 44/80 (55.2%); p = 0.037]. On the other hand, the proportion of heterozygosis in rs1059476 was significantly lower in non-cirrhotic than in cirrhotic patients in a global analysis [56/257 (21.8%) vs 26/80 (32.5%); p = 0.038] and also when we compared low vs high fibrosis [42/206 (20.4%) vs 40/131 (32.1%); p = 0.024]. Furthermore, the presence of allele T was significantly higher in high vs low fibrosis [45/131 (34.4%) vs 47/ 206 (22.8%); p = 0.015]. No correlation between HCV-genotype or subtype and LFP was found. In vitro analysis comparing the presence of alleles C or T in rs1059476 showed that kinase activity and interaction with the viral protein core were significantly lower with allele T. These differences could explain a worse prognosis in presence of T allele associated to higher fibrosis stage and cirrhosis. Conclusions: These results suggest that both alleles C at rs1059476 and T at rs2241909 positions of AURKB can have a protective effect in LFP in CHC patients, and confirm the important role of the HCV induced epigenetic modification in the progression of liver disease. The combined analysis of SNPs from different genes implicated in the HCV control of epigenetic marks could have clinical value as prognostic marker of LFP. Reference [1]
J Hepatol 2015; 63:312–9.
THU-295 Spatiotemporal functional genomics uncovers cell circuits triggering liver disease biology of hepatitis C virus infection J. Lupberger1,2, T. Croonenborghs3,4, M. Jovanovic3, P. Mertins3, N. Van Renne1,2, S. Bandiera1,2, H.E. Sagire2,5, C. Thumann1,2, J. Kozubek3,4, K. Kiani3,4, N. Bardeesy6, Y. Hoshida7, S. Carr3, A. Regev3, M.B. Zeisel1,2, N. Pochet3,4, T.F. Baumert1,2,8. 1INSERM U1110; 2Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg, Strasbourg, France; 3Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge; 4Program in Translational NeuroPsychiatric Genomics, Brigham and Women’s Hospital, Harvard Medical School, Boston, United States; 5INSERM U1110, Université de Strasbourg, Strasbourg, France; 6Cancer Center, Massachussetts General Hospital, Harvard Medical School, Boston; 7 Division of Liver Diseases, Department of Medicine, Liver Cancer Program, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States; 8Pôle Hépato-Digestif, Institut HospitaloUniversitaire, Hôpitaux Universitaires de Strasbourg, Strasbourg, France E-mail: [email protected] S320
Background and Aims: While many pathways of HCV-host interactions have been characterized in detail, a systematic understanding of how HCV establishes infection and causes disease is still only partially understood. To address this question, we probed the functional genomic architecture of persistent HCV infection within the host cell over time. Methods: Using re-differentiated hepatocyte-like Huh7.5.1 cells infected with purified HCVcc, we performed bulk and single cell RNA sequencing, proteomics and metabolomics, and we studied the kinetics of 21,950 mRNAs and 8,297 proteins (7,537 uniquely mapped overlapping genes) using integrative functional genomic analyses. Specifically, we enhanced advanced computational approaches for the integrated analysis of these profiles with known functional categories (MSigDB), protein-protein interaction networks (BioPlex), and genetic perturbation studies (LINCS). Results: Our analyses identified genes in major known functional categories being altered by HCV infection. Among others we revealed a marked and significant modulation of gene sets for metabolism and hepatocarcinogenesis. Alteration of glucose-, bile acid- and lipid metabolism was accompanied by a Warburg-like shift in metabolism. An elevated oncogenic pressure was evident by markedly impaired G2/M checkpoint regulation, DNA repair and alterations of chromatin remodeling, factors of epithelial to mesenchymal transition and telomere homeostasis. Moreover, distinct differences between transcriptome and proteome emphasize the relevance of pre- and posttranslational regulatory mechanisms modulated by HCV infection. Conclusions: Our integrative analyses provide a comprehensive and unique atlas of the cellular processes manipulated and triggered by HCV infection. These analyses contribute to uncover how HCV perturbs host cell circuits to enable viral persistence and drives chronic liver disease and hepatocarcinogenesis. Our spatiotemporal functional genomics analysis provides a unique opportunity to discover of novel drivers for virus-induced liver disease including metabolic disorders and cancer. THU-296 Hepatocyte single cell sequencing to identify transcript modulation by hepatitis C virus J.D. Debes1, C. Reilly2, G. van Oord3, P.R. Bohjanen4, T. vanwolleghem3, A. Boonstra3. 1Medicine/Gastroenterology/ID; 2Biostatistics, University of Minnesota, Minneapolis, United States; 3Gastroenterology, Erasmus MC, Rotterdam, The Netherlands; 4Infectious Disease, University of Minnesota, Minneapolis, United States E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) is an enveloped single-stranded RNA virus that uses the human gene expression machinery to facilitate viral replication through mechanisms that are incompletely understood. HCV infects a partial number of hepatocytes in each infected individual, and the specific gene-modulation effects of the virus in each hepatocyte remain unknown. Methods: We evaluated transcript-changes in HCV-infected hepatocytes from HCV-infected patients. Two HCV-infected individuals underwent needle biopsy. Following biopsy, samples were processed via fluorescence activated cell sorting (FACS) in which staining for specific antibodies allowed us to gate different cells types and isolate single hepatocytes as well as clusters of hepatocytes in a single well. These hepatocytes (both individual and clusters) underwent RNA extraction and cDNA formation and were subjected to RNASeq analysis using Illumina platform. Results: We were able to analyze 11 samples from the 2 patients, comprised of single hepatocytes as well as hepatocyte clusters. Each sample revealed a number of transcripts that varied from 61 to 15525. All samples matched positive for albumin ( present only in hepatocytes) and several transcripts were related to immune responses (FoxP1, B2M, HSP90AB1) as well as cell proliferation (APOA1, NASP).
Journal of Hepatology 2017 vol. 66 | S95–S332
POSTER PRESENTATIONS Samples were mapped to the HCV genome to identify hepatocytes infected with HCV. We found 9 samples that mapped the HCV genome and 2 that did not. Interestingly, several transcripts related to cancer development, such as GSTP1, VISP and KEAP1 were only present in the HCV-infected hepatocytes. Conclusions: We successfully identified a method to isolate single hepatocytes and address transcript modulation via RNASeq. This approach will provide invaluable information about HCV infection as well as cancer-related mechanisms and could help identify patients at risk for liver cancer following treatment for HCV. THU-297 Multiple ascending dose study of seraprevir potassium, a nonstructural protein 3/4A replication complex inhibitor, in patients infected with hepatitis C virus Y. Ding1, H. Zhang1, J. Niu1, H. Chen1, Y. Hu1, X. Li1, X. Zhu1, J. Liu1. 1Phase I Clinical Research Center, The First Hospital of Jilin University, Changchun, China E-mail: [email protected] Background and Aims: The antiviral activity, resistance profile, pharmacokinetics (PK), safety, and tolerability of seraprevir potassium, a second generation nonstructural protein 3/4A (NS3/4A) replication complex inhibitor, were evaluated in a double-blind, placebo-controlled, sequential panel, multiple ascending dose study. Methods: Forty patients with chronic hepatitis C virus (HCV) infection were randomized to receive a 3.5-day course of seraprevir potassium (200 mg BID, 400 mgQD, 400 mgBID, 100 mgBID, 200 mgQD) or placebo in a ratio of 3:1 after meal. The antiviral activity, resistance profile, pharmacokinetics (PK), safety, and tolerability were assessed in the study. Results: The mean decline from baseline in HCV RNA were −3.35, −2.52, −3.32, −3.38, −3.57 log10 IU/mL at 100 mg BID, 200 mg QD, 200 mg BID, 400 mg QD, 400 mg BID with hepatitis c virus genotype 1 patients respectively. the placebo group showed no evidence of antiviral activity. Some patients experienced viral rebound on or before last treatment with seraprevir potassium monotherapy. viral rebound or non-respons were associated with viral variants that had been previously implicated in resistance development in the in vitro replicon system. The PK profile was supportive of twice-daily dosing with median peak plasma concentrations at 2.33–3.5 hours postdose and mean terminal half-life of 4.29–6.14 hours after last dosage. Steady state was achieved following 3 days of daily dosing. The accumulation rate was similar among the diferent groups (1.0–1.18), which indicates that the accumulation is low. The relationship between Seraprevir Potassium exposures (AUC and Cmax) and dosage were not linear and the slopes were 2.3 and 2.24 respectively. seraprevir potassium was well tolerated in all dose groups, with adverse events occurring with a similar frequency in seraprevir potassium – and placebo-treated groups, Such as nausea, vomit and headache. There were no significant clinically relevant changes in vital signs, laboratory, or electrocardiogram parameters. Conclusions: The second generation NS3/4A replication complex inhibitor, seraprevir potassium is well tolerated and produced a rapid and substantial decrease in HCV-RNA levels in patients chronically infected with HCV. On the basis of these results, seraprevir potassium 100 and 200 mg BID, were recommended for further evaluation in phase II trials. THU-298 Inosine triphosphate pyrophosphatase enhances the effect of ribavirin on hepatitis C virus cell culture infection K. Nyström1, G. Pettersson1, P.H. Wanrooij2, S. Brunet1, J. Said1, G. Ortolani1, J. Waldenström1, L. Adamek1, K.-W. Tang1, P. Norberg1, A. Chabes2, K. Hellstrand1, H. Norder1, M. Lagging1. 1Department of Infectious Diseases, University of Gothenburg, Gothenburg; 2Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden E-mail: [email protected]
Background and Aims: Genetic variants of the inosine triphosphate pyrophosphatase gene (ITPA), resulting in decreased enzymatic activity of the corresponding enzyme, ITPase, are known to correlate with a decreased risk of ribavirin-induced anemia, but are also associated with an increased SVR in patients treated with peginterferon-alpha and ribavirin. As both ITPase and ribavirin are involved in the nucleotide salvage pathway and reduced risk of relapse after treatment of hepatitis C, we have investigated the effect of ITPase activity and ribavirin treatment of HCVcc infection of hepatocytes. Methods: Hepatocytes were transfected with ITPA siRNA or negative control siRNA. The cells were then treated with various concentrations of ribavirin and infected with HCVcc. The cells were analysed for progeny virus production and viral RNA replication. Intracellular nucleotide triphosphate levels were measured with high performance liquid chromatography (HPLC). Deep sequencing of the viral genome was performed to characterize mutations after ITPA siRNA and ribavirin treatment compared to controls. Results: siRNA inhibition of ITPA expression together with ribavirin treatment of cells infected with HCVcc resulted in significantly reduced virus RNA expression and progeny virus production compared to negative control siRNA using 100 or 200 μM ribavirin. HPLC demonstrated GTP depletion in both ITPA or negative control siRNA transfected cells treated with 100 or 200 μM ribavirin, though ITPA siRNA transfected cells contained significantly higher amounts of ribavirin triphosphate (RTP) as compared to negative control siRNA transfected cells. Deep sequencing of viral RNA from the infected cells demonstrated an increase in single nucleotide variations in samples inhibited with ITPA siRNA compared to negative control siRNA using 100 and 200 μM ribavirin treatment, primarily from C-to-U and G-toA, as has previously been demonstrated as ribavirin-induced mutagenesis. Conclusions: Reduced expression of ITPA appears to augment the effect of ribavirin on HCV replication, increased intracellular RTP levels resulting in increased mutagenesis. These data indicate that ITPA possibly is able to dephosphorylate RTP to ribavirin monophosphate. This may imply that ribavirin dosing should be tailored also based on ITPA genotype. Additionally, as ribavirin has anti-viral activity against many other viruses including hepatitis E virus, ITPA may be a novel antiviral target in addition to possibly contributing to innate immunity. THU-299 Identification of glycogen synthase kinase 3 beta as a novel host factor of hepatitis C virus replication M. Saleh1, S. Rüschenbaum1, C. Welsch1, S. Zeuzem1, D. Moradpour2, J. Gouttenoire2, C.M. Lange1. 1Department of Internal Medicine 1, Goethe university hospital Frankfurt, Frankfurt a.M., Germany; 2Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules of these metabolic pathways. Yet, the role of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism and insulin signaling, has not been investigated in the HCV life cycle. Methods: The impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring HCV subgenomic genotype 1b replicons or infected with cell-culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in the life cycle of hepatitis E virus (HEV) was investigated to assess virus specificity of the observed findings.
Journal of Hepatology 2017 vol. 66 | S95–S332
S321
J Hepatol 2015; 63:312–9.
THU-295 Spatiotemporal functional genomics uncovers cell circuits triggering liver disease biology of hepatitis C virus infection J. Lupberger1,2, T. Croonenborghs3,4, M. Jovanovic3, P. Mertins3, N. Van Renne1,2, S. Bandiera1,2, H.E. Sagire2,5, C. Thumann1,2, J. Kozubek3,4, K. Kiani3,4, N. Bardeesy6, Y. Hoshida7, S. Carr3, A. Regev3, M.B. Zeisel1,2, N. Pochet3,4, T.F. Baumert1,2,8. 1INSERM U1110; 2Institut de Recherche sur les Maladies Virales et Hépatiques, Université de Strasbourg, Strasbourg, France; 3Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge; 4Program in Translational NeuroPsychiatric Genomics, Brigham and Women’s Hospital, Harvard Medical School, Boston, United States; 5INSERM U1110, Université de Strasbourg, Strasbourg, France; 6Cancer Center, Massachussetts General Hospital, Harvard Medical School, Boston; 7 Division of Liver Diseases, Department of Medicine, Liver Cancer Program, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States; 8Pôle Hépato-Digestif, Institut HospitaloUniversitaire, Hôpitaux Universitaires de Strasbourg, Strasbourg, France E-mail: [email protected] S320
Background and Aims: While many pathways of HCV-host interactions have been characterized in detail, a systematic understanding of how HCV establishes infection and causes disease is still only partially understood. To address this question, we probed the functional genomic architecture of persistent HCV infection within the host cell over time. Methods: Using re-differentiated hepatocyte-like Huh7.5.1 cells infected with purified HCVcc, we performed bulk and single cell RNA sequencing, proteomics and metabolomics, and we studied the kinetics of 21,950 mRNAs and 8,297 proteins (7,537 uniquely mapped overlapping genes) using integrative functional genomic analyses. Specifically, we enhanced advanced computational approaches for the integrated analysis of these profiles with known functional categories (MSigDB), protein-protein interaction networks (BioPlex), and genetic perturbation studies (LINCS). Results: Our analyses identified genes in major known functional categories being altered by HCV infection. Among others we revealed a marked and significant modulation of gene sets for metabolism and hepatocarcinogenesis. Alteration of glucose-, bile acid- and lipid metabolism was accompanied by a Warburg-like shift in metabolism. An elevated oncogenic pressure was evident by markedly impaired G2/M checkpoint regulation, DNA repair and alterations of chromatin remodeling, factors of epithelial to mesenchymal transition and telomere homeostasis. Moreover, distinct differences between transcriptome and proteome emphasize the relevance of pre- and posttranslational regulatory mechanisms modulated by HCV infection. Conclusions: Our integrative analyses provide a comprehensive and unique atlas of the cellular processes manipulated and triggered by HCV infection. These analyses contribute to uncover how HCV perturbs host cell circuits to enable viral persistence and drives chronic liver disease and hepatocarcinogenesis. Our spatiotemporal functional genomics analysis provides a unique opportunity to discover of novel drivers for virus-induced liver disease including metabolic disorders and cancer. THU-296 Hepatocyte single cell sequencing to identify transcript modulation by hepatitis C virus J.D. Debes1, C. Reilly2, G. van Oord3, P.R. Bohjanen4, T. vanwolleghem3, A. Boonstra3. 1Medicine/Gastroenterology/ID; 2Biostatistics, University of Minnesota, Minneapolis, United States; 3Gastroenterology, Erasmus MC, Rotterdam, The Netherlands; 4Infectious Disease, University of Minnesota, Minneapolis, United States E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) is an enveloped single-stranded RNA virus that uses the human gene expression machinery to facilitate viral replication through mechanisms that are incompletely understood. HCV infects a partial number of hepatocytes in each infected individual, and the specific gene-modulation effects of the virus in each hepatocyte remain unknown. Methods: We evaluated transcript-changes in HCV-infected hepatocytes from HCV-infected patients. Two HCV-infected individuals underwent needle biopsy. Following biopsy, samples were processed via fluorescence activated cell sorting (FACS) in which staining for specific antibodies allowed us to gate different cells types and isolate single hepatocytes as well as clusters of hepatocytes in a single well. These hepatocytes (both individual and clusters) underwent RNA extraction and cDNA formation and were subjected to RNASeq analysis using Illumina platform. Results: We were able to analyze 11 samples from the 2 patients, comprised of single hepatocytes as well as hepatocyte clusters. Each sample revealed a number of transcripts that varied from 61 to 15525. All samples matched positive for albumin ( present only in hepatocytes) and several transcripts were related to immune responses (FoxP1, B2M, HSP90AB1) as well as cell proliferation (APOA1, NASP).
Journal of Hepatology 2017 vol. 66 | S95–S332
POSTER PRESENTATIONS Samples were mapped to the HCV genome to identify hepatocytes infected with HCV. We found 9 samples that mapped the HCV genome and 2 that did not. Interestingly, several transcripts related to cancer development, such as GSTP1, VISP and KEAP1 were only present in the HCV-infected hepatocytes. Conclusions: We successfully identified a method to isolate single hepatocytes and address transcript modulation via RNASeq. This approach will provide invaluable information about HCV infection as well as cancer-related mechanisms and could help identify patients at risk for liver cancer following treatment for HCV. THU-297 Multiple ascending dose study of seraprevir potassium, a nonstructural protein 3/4A replication complex inhibitor, in patients infected with hepatitis C virus Y. Ding1, H. Zhang1, J. Niu1, H. Chen1, Y. Hu1, X. Li1, X. Zhu1, J. Liu1. 1Phase I Clinical Research Center, The First Hospital of Jilin University, Changchun, China E-mail: [email protected] Background and Aims: The antiviral activity, resistance profile, pharmacokinetics (PK), safety, and tolerability of seraprevir potassium, a second generation nonstructural protein 3/4A (NS3/4A) replication complex inhibitor, were evaluated in a double-blind, placebo-controlled, sequential panel, multiple ascending dose study. Methods: Forty patients with chronic hepatitis C virus (HCV) infection were randomized to receive a 3.5-day course of seraprevir potassium (200 mg BID, 400 mgQD, 400 mgBID, 100 mgBID, 200 mgQD) or placebo in a ratio of 3:1 after meal. The antiviral activity, resistance profile, pharmacokinetics (PK), safety, and tolerability were assessed in the study. Results: The mean decline from baseline in HCV RNA were −3.35, −2.52, −3.32, −3.38, −3.57 log10 IU/mL at 100 mg BID, 200 mg QD, 200 mg BID, 400 mg QD, 400 mg BID with hepatitis c virus genotype 1 patients respectively. the placebo group showed no evidence of antiviral activity. Some patients experienced viral rebound on or before last treatment with seraprevir potassium monotherapy. viral rebound or non-respons were associated with viral variants that had been previously implicated in resistance development in the in vitro replicon system. The PK profile was supportive of twice-daily dosing with median peak plasma concentrations at 2.33–3.5 hours postdose and mean terminal half-life of 4.29–6.14 hours after last dosage. Steady state was achieved following 3 days of daily dosing. The accumulation rate was similar among the diferent groups (1.0–1.18), which indicates that the accumulation is low. The relationship between Seraprevir Potassium exposures (AUC and Cmax) and dosage were not linear and the slopes were 2.3 and 2.24 respectively. seraprevir potassium was well tolerated in all dose groups, with adverse events occurring with a similar frequency in seraprevir potassium – and placebo-treated groups, Such as nausea, vomit and headache. There were no significant clinically relevant changes in vital signs, laboratory, or electrocardiogram parameters. Conclusions: The second generation NS3/4A replication complex inhibitor, seraprevir potassium is well tolerated and produced a rapid and substantial decrease in HCV-RNA levels in patients chronically infected with HCV. On the basis of these results, seraprevir potassium 100 and 200 mg BID, were recommended for further evaluation in phase II trials. THU-298 Inosine triphosphate pyrophosphatase enhances the effect of ribavirin on hepatitis C virus cell culture infection K. Nyström1, G. Pettersson1, P.H. Wanrooij2, S. Brunet1, J. Said1, G. Ortolani1, J. Waldenström1, L. Adamek1, K.-W. Tang1, P. Norberg1, A. Chabes2, K. Hellstrand1, H. Norder1, M. Lagging1. 1Department of Infectious Diseases, University of Gothenburg, Gothenburg; 2Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden E-mail: [email protected]
Background and Aims: Genetic variants of the inosine triphosphate pyrophosphatase gene (ITPA), resulting in decreased enzymatic activity of the corresponding enzyme, ITPase, are known to correlate with a decreased risk of ribavirin-induced anemia, but are also associated with an increased SVR in patients treated with peginterferon-alpha and ribavirin. As both ITPase and ribavirin are involved in the nucleotide salvage pathway and reduced risk of relapse after treatment of hepatitis C, we have investigated the effect of ITPase activity and ribavirin treatment of HCVcc infection of hepatocytes. Methods: Hepatocytes were transfected with ITPA siRNA or negative control siRNA. The cells were then treated with various concentrations of ribavirin and infected with HCVcc. The cells were analysed for progeny virus production and viral RNA replication. Intracellular nucleotide triphosphate levels were measured with high performance liquid chromatography (HPLC). Deep sequencing of the viral genome was performed to characterize mutations after ITPA siRNA and ribavirin treatment compared to controls. Results: siRNA inhibition of ITPA expression together with ribavirin treatment of cells infected with HCVcc resulted in significantly reduced virus RNA expression and progeny virus production compared to negative control siRNA using 100 or 200 μM ribavirin. HPLC demonstrated GTP depletion in both ITPA or negative control siRNA transfected cells treated with 100 or 200 μM ribavirin, though ITPA siRNA transfected cells contained significantly higher amounts of ribavirin triphosphate (RTP) as compared to negative control siRNA transfected cells. Deep sequencing of viral RNA from the infected cells demonstrated an increase in single nucleotide variations in samples inhibited with ITPA siRNA compared to negative control siRNA using 100 and 200 μM ribavirin treatment, primarily from C-to-U and G-toA, as has previously been demonstrated as ribavirin-induced mutagenesis. Conclusions: Reduced expression of ITPA appears to augment the effect of ribavirin on HCV replication, increased intracellular RTP levels resulting in increased mutagenesis. These data indicate that ITPA possibly is able to dephosphorylate RTP to ribavirin monophosphate. This may imply that ribavirin dosing should be tailored also based on ITPA genotype. Additionally, as ribavirin has anti-viral activity against many other viruses including hepatitis E virus, ITPA may be a novel antiviral target in addition to possibly contributing to innate immunity. THU-299 Identification of glycogen synthase kinase 3 beta as a novel host factor of hepatitis C virus replication M. Saleh1, S. Rüschenbaum1, C. Welsch1, S. Zeuzem1, D. Moradpour2, J. Gouttenoire2, C.M. Lange1. 1Department of Internal Medicine 1, Goethe university hospital Frankfurt, Frankfurt a.M., Germany; 2Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland E-mail: [email protected] Background and Aims: Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules of these metabolic pathways. Yet, the role of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism and insulin signaling, has not been investigated in the HCV life cycle. Methods: The impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring HCV subgenomic genotype 1b replicons or infected with cell-culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in the life cycle of hepatitis E virus (HEV) was investigated to assess virus specificity of the observed findings.
Journal of Hepatology 2017 vol. 66 | S95–S332
S321