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Simultaneous detection of hepatitis C virus and interferon stimulated gene expression in infected human liver

Stefan F Wieland1, Zuzanna Makowska2, Benedetta Campana2, 3, Diego Calabrese2, Michael T. Dill2, 3, Josan Chung1, Francis V. Chisari1, Markus H. Heim2, 3
1Department of Molecular and Experimental Medicine, The Scripps Research Institute/ La Jolla, CA; 2Department of Biomedicine, University of Basel, Basel, Switzerland; 3Department of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland

Background: Over 20 years after the molecular cloning and identification of hepatitis C virus (HCV) a reproducible method to identify HCV infected hepatocytes in human liver biopsies is still lacking and this has been a major obstacle for understanding host-virus interactions in HCV infections. Methods: We adapted an in situ hybridization (ISH) system (QuantiGene® ViewRNA, Affymetrix, Santa Clara, CA) using HCV isolate specific probes. Snap frozen liver biopsies of 18 patients with chronic hepatitis C (CHC), different viral genotypes and a wide range of serum viral loads were analysed. For each biopsy, HCV RNA was isolated and sequenced, and highly specific probe sets were designed. We further developed the method using multiplex ISH to simultaneously detect HCV and interferon stimulated gene expression. Results: The system had an extremely high signal to noise ratio with virtually no unspecific background staining and no cross-reactivity by probe sets targeting plus or minus strand HCV RNA, different viral RNAs, and even between HCV isolates with identical genotypes, we found only weak cross-reactivity. The average percentage of infected hepatocytes in the 18 biopsies ranged from 1.3% to 53.9%. There was a significant positive correlation between the proportion of infected hepatocytes and the viral load in the serum and the liver, but not with the HCV genotype. HCV positive cells occurred in clusters. A quantitative analysis of the spatial relationship between HCV RNA and interferon stimulated gene (ISG) mRNA expression in the subset of patients with an induced endogenous IFN system revealed a significant correlation. ISG signal intensity was lowest in uninfected cells with uninfected neighbors, intermediate in uninfected cells with at least one HCV positive neighbor, and highest in HCV positive cells. Conclusion: Over 20 years after the cloning and identification of HCV, we have developed the first highly sensitive, specific and reproducible in situ detection systems that allows to identify HCV infected cells in liver biopsies in patients with viral loads as low as 1 0E4 IU/ml. A quantitative analysis of the number and spatial distribution of HCV infected cells and ISG expression revealed a number of fundamental question concerning HCV-host interactions: HCV infects only hepatocytes. The percentage of infected hepatocytes varies from 1 to 54 %, and correlates with serum viral load. HCV infected cells appear in clusters, favoring a model of cell to cell transmission. Finally, the positive correlation of HCV RNA signals with ISG mRNA expression in patients with induced ISG expression reveals that HCV is the central driver of ISG induction.

Disclosures:

The following people have nothing to disclose: Stefan F Wieland, Zuzanna Makowska, Benedetta Campana, Diego Calabrese, Michael T. Dill, Josan Chung, Francis V. Chisari, Markus H. Heim

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Persistently Infected Hepatitis C Virus Cell Culture Impairs Type I But Not The Type III IFN Signaling

Partha K. Chandra1,Kyoungsub Song1, Darren P. Baker2, Shuonghu Liu3, Curt H. Hagedorn3, Serge Y .Fuchs4, Nathan J. Shores5, Tong Wu1, Luis A. Balart5, Srikanta Dash1
1Pathalogy and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA; 2Biogen Idec Inc., Cambridge, MA; 3Medicine, University of Utah School of Medicine, Salt Lake City, UT; 4Animal Biology, University of Pennsylvania, Philadelphia, PA; 5Gastroenetrology and Hepatology, Tulane University School of Medicine, New Orleans, LA

Background: Combination therapy using peg-interferon (IFNa), ribavirin (RBV) and protease inhibitor has improved the sustained antiviral response of chronic hepatitis C virus (HCV)1a infection. However, the treatment response has not improved significantly among patients who are prior non-responders to IFN-a and RBV and the mechanism of HCV resistance is not well understood. Aim: A persistent HCV replication cell culture model was developed to examine the impact of high-level viral replication on IFN-α and RBV treatment induced viral clearance. Methods: A persistent HCV infected cell culture model was established by using pJFH-delta V3-Rluc clone. HCV replication was confirmed by measuring the core and NS5A-Rluc protein expression by Western blotting and Renilla luciferase activity. Endoplasmic reticulum (ER) stress response due to HCV infection was measured by measuring ATF6 Firefly luciferase activity and autophagy induction was confirmed by measuring LC3II, p62 and Beclin 1 protein levels by Western blotting and immunocytochemical staining. The modulation of the expression of IFN-receptors, Jak-Stat signaling pathway and RBV pump activity were observed by Western blotting, FACS-analysis and RBV uptake assay. Results: Persistently HCV-infected culture produces a high titer virus and shows an impaired antiviral response to IFN-a plus RBV combination treatment. IFN-λ shows a strong and sustained antiviral response and clears HCV replication to a completion. HCV replication induced ER-stress and an autophagy response that selectively down regulated IFN-a receptor-1 chain (IFNAR1) of the type I, but not the type II, or type ||| IFN-receptors. Down regulated expression of IFNAR1 resulted in defective Jak-Stat signaling, impaired Stat-phosphorylation, and nuclear translocation. Furthermore, HCV replication impaired RBV uptake due to reduced expression of the nucleoside transporters ENT1 and CNT1.Chemically induced ER-stress and autophagy response selectively down regulated IFNAR1, but not the IFNγR1 (IFN-γ receptor) or IL10Rβ (IFN-γ receptor) receptors. Silencing ER stress and autophagy response using chemical inhibitors or by siRNAs additively inhibited HCV replication and induced viral clearance by IFNα plus RBV treatment. Conclusions: Our results suggest that HCV induced ER-stress and the autophagy response selectively impairs type I, but not type ||| IFN signaling, which is why IFNγ showed a sustained antiviral response against HCV infection. Inhibiting ER stress and the autophagy response overcome IFN-α plus RBV resistance mechanisms associated with HCV infection. Acknowledgement: The work was supported by NIH grants CA127481 and CA089121.

Disclosures:

Darren P. Baker - Employment: Biogenldec; Stock Shareholder: Biogenldec

Nathan J. Shores - Advisory Committees or Review Panels: Gilead; Speaking and Teaching: Vertex, Merck, Salix

Luis A. Balart - Advisory Committees or Review Panels: Genentech, Genentech; Grant/Research Support: Merck, Genentech, Bayer, conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, Merck, Genentech, Bayer, Conatus, Ocera, Hyperion, Gilead Sciences, Bristol Myers Squibb, Mochida, Eisai, Vertex, takeda, GI Dynamics; Speaking and Teaching: Merck, Merck, Merck, Merck

The following people have nothing to disclose: Partha K. Chandra, Kyoungsub Song, Shuanghu Liu, Curt H. Hagedorn, Serge Y. Fuchs, Tong Wu, Srikanta Dash

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Expression levels of IFNL4 and other IFN-λs are determined by dinucleotide polymorphism of IFNL4 and correlate with basal expression levels of interferon stimulated genes and effect of interferon

Hiromi Abe1, 2, C. Nelson Hayes1, 2,Nobuhiko Hiraga1, Michio Imamura1, Daiki Miki1, 2, Masataka Tsuge1, Hidenori Ochi1, 2, Kazuaki Chayama1, 2
1Hiroshima University, Hiroshima, Japan; 2RIKEN Institute, Hiroshima, Japan

Background and aim: Recently, it has been reported that a dinucleotide polymorphism (ss469415590, ΔG/TT) is closely related with presence of IFNL4 in hepatocytes and is associated with the effect of IFN and spontaneous clearance rate of hepatitis C virus. We previously reported that there is reciprocal control between expression levels of anti-viral effecter genes and negative regulator of interferon stimulated genes by IL28B gene polymorphism. The aim of this study is to investigate the relationship between IFNL4 polymorphism and expression level of IFNL4 as well as other IFN-λs and ISGs in the liver and their correlation with effect of IFN. Method: Dinucleotide polymorphism of IFNL4 (ss469415590, ΔG or TT) was determined by Invader assay. Expression levels of IFNL4 and IFN-λs were determined by TaqMan assay and digital PCR by designing TaqMan probe and primers targeting exon2 and exon3 of IFNL4.Expression levels of these genes were compared with those of ISGs and effect of IFN treatment in patients with chronic hepatitis C. Results: Genotyping data showed that the IFNL4 polymorphism ss469416690 was tightly linked with the rs8099917 SNP near the IFNL3 (IL28B) locus. Expression of IFNL4 in liver biopsies from both ΔG/ΔG and TT/TT genotypes were detectable with digital PCR, although IFNL4 expression level was very low. The expression level of IFNL4 was significantly higher in ss469416690 ΔGΔG and ΔG/TT patients than that in TT/TT patients (P<0.009). Expression levels and IFN-λ and other antiviral interferon stimulated genes such as MxA, 〇AS1, PKR were also significantly lower in IFNL4 SNP TT/TT patients. We further analyzed levels of IFNL4 expression in livers of HCV infected humanized uPA-SCID mice transplanted with human hepatocytes with both ss469416690 ΔGΔG and TT/TT human hepatocytes transplanted mice. The expression level of IFNL4 in mice transplanted with ss469416690 ΔGΔG hepatocytes was about 10 times higher than in mice transplanted with ss469416690 TT/TT hepatocytes (P<0.05). Interestingly, the expression level of IFNL4 was reduced to 1/3 by IFN-α treatment, and reduced to 1/10 by IL-28B treatment in ss469416690 ΔGΔG mice hepatocytes. In contrast, the expression level of IFNL4 was only slightly reduced by IFN-γ treatment, and reduced to 1 / 7 by I L-2 8 B treatment in ss469416690 TT/TT mice hepatocytes. Conclusion: There are significant differences in basal IFNL4, IFN-γs and other antiviral interferon stimulated genes associated with the IFNL4 polymorphism. Higher expression levels of IFNL4 might be a reason for the dampened ISG expression response after IFN-α administration and the poor response to IFN-a therapy.

Disclosures:

Kazuaki Chayama - Consulting: Abbvie; Grant/Research Support: Dainippon Sumitomo, Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, Toray, BMS, MSD; Speaking and Teaching: Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, KYORIN, Nihon Medi-Physics, BMS, Dainippon Sumitomo, MSD, ASKA, Astellas, AstraZeneca, Eisai, Olympus, GlaxoSmithKline, ZERIA, Bayer, Minophagen,

JANSSEN, JIMRO, TSUMURA, Otsuka, Taiho, Nippon Kayaku, Nippon

Shinyaku, Takeda, AJINOMOTO, Meiji Seika, Toray

The following people have nothing to disclose: Hiromi Abe, C. Nelson Hayes, Nobuhiko Hiraga, Michio Imamura, Daiki Miki, Masataka īsuge, Hidenori Ochi

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Identification of Viral Double-stranded RNA as a Substrate for Hepatitis C Relapse and as a Target for Ribavirin

Arielle L. Klepper, Francis J. Eng, Adeeb Rahman, Brannon Weeks, Ahmed El-shamy/ M. Isabel Fiel, Gonzolo Carrasco, Sason Roayaie, Meena Bansal, Thomas D. Schiano, Andrea D. Branch
Mount Sinai School of Medicine, New York, NY

Background: One of the most enduring mysteries about HCV has been its ability to persist at undetectable levels during antiviral treatment only to reemerge. Ribavirin (RBV) reduces relapse and is used with both interferon and with direct acting antiviral drugs. The impact of RBV on HCV double-stranded RNA (dsRNA), a non-replicating form that may mediate relapse, is unknown. Methods: HCV RNAs were quantified in extracts of human liver (n=5) and in a cell culture model in which Huh-7.5 cells replicating Con1/JFH virus were treated with HCV inhibitors: peg-IFNα (IFN; 3 IU/mL, 9 IU/mL), RBV (10 μg/mL), and 2'c-methyl adenosine (2'CMA; 2.2 μM). To allow HCV dsRNA detection, samples were heated to 106°C to denature duplexes prior to qPCR. Controls were carried out with RNase III. HCV NS5A protein and dsRNA were quantified by FACS using specific antibodies. Results: HCV dsRNA was the most abundant form of HCV RNA in patient livers, accounting for about 80% of the total. HCV dsRNA titers in human liver

correlated with induction of IFIT1 (r=0.997, p<0.0005). In Huh7.5 cells, IFN caused a dose-dependent reduction in HCV ssRNA, the actively replicating form, and an increase in HCV dsRNA, the proposed viral reservoir. Changes in HCV RNA levels were measured using qRT-PCR assays targeting the HCV (+) strand 5' UTR (p<0.005), the 3'UTR (p<0.05), and the (-) strand 3' UTR (p<0.001). IFN increased the percentage of cells where HCV was in a non-replicative state, characterized by staining for dsRNA with no detectable NS5A protein (p<0.01). Of great interest, RBV did not increase HCV dsRNA. In fact, it decreased the percentage of dsRNA positive/NS5A negative cells. In keeping with clinical data showing that RBV reduces relapse, the addition of RBV to 9 IU/mL IFN reduced the ratio of HCV dsRNA: ssRNA by a factor of 2.5.It dramatically reduced the percentage of dsRNA positive/NS5A negative cells, and increased the percentage of dsRNA negative/NS5A positive cells. The HCV polymerase inhibitor, 2'CMA, was then tested. Of potential importance for anti-viral drug development, 2' CMA had effects similar to IFN, increasing HCV dsRNA and the percentage of dsRNA positive/NS5A negative cells. Conclusions: Our data suggest that HCV escapes both natural immune clearance mechanisms and IFN treatment by synthesizing viral dsRNA and entering quiescent survival mode. Consistent with this, HCV dsRNA was predominant in human livers and its levels correlated with IFIT1, a cytokine associated with IFN treatment failure. An RNA polymerase inhibitor triggered dsRNA production. In contrast, RBV, a drug used to prevent relapse, blocked production of HCV dsRNA (DA031095, DK090317).

Disclosures:

Andrea D. Branch - Grant/Research Support: Kadmon, Gilead, Janssen

The following people have nothing to disclose: Arielle L. Klepper, Francis J. Eng, Adeeb Rahman, Brannon Weeks, Ahmed El-Shamy, M. Isabel Fiel, Gonzalo Carrasco, Sasan Roayaie, Meena Bansal, Thomas D. Schiano

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EFTUD2 regulates hepatitis C virus replication through the RIG-I/MDA5 pathway

Chuanlong Zhu, Jian Hong, Lei Zhao, Pattranuch Chusri, Nikolaus Jilg, Dahlene N. Fusco, Esperance A. Schaefer, Cynthia Brisac, Stephane Chevaliez, Daniel Wambua, Lee F. Peng, Wenyu Lin, Raymond T. Chung
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA

Background/Aims: In a previous siRNA screen, we identified 22 genes that mediate IFN's antiviral effects against HCV. Among these IFN effector genes, we identified elongation factor Tu GTP binding domain containing 2 (EFTUD2), a component of the spliceosome. We hypothesized that EFTUD2 exerts its anti-HCV activity by altering expression of viral RNA sensor molecules, including RIG-1 and MDA5.We evaluated this hypothesis using Huh7.5.1 cells, which are RIG-I pathway signaling defective and more permissive for HCV infection compared to their parental Huh7 cells. Methods: We performed siRNA knockdown of EFTUD2, RIG-I, or MDA5 in uninfected or JFH1-infected Huh7.5.1 or Huh7 cells. Selected cells were incubated with the RIG-I-like receptor (RLR) signaling inhibitor BX795.Effects on IFN signaling were monitored by a luciferase reporter system driven by ISRE. Selected gene mRNA levels and HCV replication were monitored by qPCR. Results: JFH1 HCV replicated more efficiently in Huh7.5.1 than in Huh7 cells (281808±13506 vs 10402±574) at 24hr JFH1 infection. Treatment with BX795 increased JFH1 HCV replication from 9918±494 to 31208±1612 (P<0.001) in Huh7 cells, but had no significant effect on HCV replication [295893±22768 (BX795) vs 249740±19938 (1%DMSO)] in Huh7.5.1 cells.

EFTUD2 siRNA increased HCV replication by 1.8- and 2.8-fold in JFH1-infected Huh7.5.1 and Huh7 cells respectively. EFTUD2 siRNA significantly decreased RIG-1 and MDA5 mRNA transcription in Huh7.5.1 and Huh7 cells. However, EFTUD2 siRNA did not affect IFNAR1 or IRF9 mRNA expression, or IFN stimulated ISRE-signaling in either Huh7.5.1 or Huh7 cells, suggesting that EFTUD2 does not regulate classical ISGs through Jak-STAT or ISRE signaling. Overexpression of EFTUD2 reduced HCV replication from 12731 ±785 to 4243±265 (P<0.001) in JFH1-infected Huh7 cells. Interestingly, BX795 abrogated EFTUD2-mediated inhibition of HCV replication [11, 406±1486 (pEFTUD2+BX795) vs 4160±532 (pEFTUD2), P=0.0013]. Overexpression of EFTUD2 more modestly inhibited JFH1 HCV replication from 302649±21437 to 226986±14577 (P=0.007) in Huh7.5.1 cells. In contrast, BX795 did not rescue the observed EFTUD2-mediated inhibition of HCV replication [260059±30564 (pEFTUD2+BX795) vs 208694±17938 (pEFTuD2), P=0.07] in these cells. Conclusions: EFTUD2 exerts its anti-HCV action primarily through regulation of the RIGI/MDA5 pathways, since overexpression of EFTUD2 suppresses HCV replication in a RIG-I competent cell line, and this suppression rescued by RLR inhibition. Conversely, EFTUD2 has no effect on Jak-STAT and ISRE signaling. These findings suggest a novel role for EFTUD2 in its interaction with the viral RNA sensor pathway.

Disclosures:

Raymond T . Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead

The following people have nothing to disclose: Chuanlong Zhu, Jian Hong, Lei Zhao, Pattranuch Chusri, Nikolaus Jilg, Dahlene N. Fusco, Esperance A. Schaefer, Cynthia Brisac, Stephane Chevaliez, Daniel Wambua, Lee F. Peng, Wenyu Lin

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Impaired IFN signaling in chronic hepatitis C with advanced fibrosis via the TGF-β signaling pathway

Takayoshi Shirasaki1 ' 2, Masao Honda12, Tetsuro Shimakami1 , Kazuhisa Murai1 , 2 , Takayuki Shiomoto1 , 2,. Hikari Okada1, Riuta Takabatake1, Stanley M. Lemon3, Seishi Murakami1, Shuichi Kaneko1
1Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Kanazawa Ishikawa, Jopon; 2Department of Advanced Medical Technology, Kanazawa University Graduate School of Health Medicine, Kanazawa Ishikawa, Japan; 3Division of Infectious Diseases, The University of North Carolina at Chapel Hill, Chapel Hill, NC

Objective: The response of chronic hepatitis C (CHC) to IFN treatment is hampered in patients with advanced liver fibrosis, and IFN might also affect the efficacy of triple therapy (PegIFN+RBV+DAA). Previous studies have shown that malnutrition impairs IFN signaling by inhibiting mT〇RC1 and activating Socs3-mediated IFN inhibitory signaling via Foxo3a. Branchedchain amino acids (BCAA) can restore impaired IFN signaling and inhibit HCV replication under conditions of malnutrition (Gastroenterology 2011). Transforming growth factor-beta (TGF-β) plays an important role in fibrosis development, but its role in IFN signaling is unclear. Here we investigate the role of TGF-β on IFN signaling in HCV replication. Methods: Gene expression profiling of 91 patients with CHC was performed using GeneChip. The replicon (H77S) was transfected into Huh7.5 cells. HCV RNA and mRNA levels of ISGs, Smad, Foxo3a, and Socs3 were evaluated by RT-PCR and Western blotting. We also evaluated promoter activation of Foxo3a and Socs3 by TGF-p. Results: Hepatic TGF-p signaling was significantly upregulated in patients with advanced fibrosis. Smad2 and Foxo3a correlated significantly in transcriptional level, and Foxo3a levels correlated significantly with Socs3 expression. In vitro expression of Foxo3a was significantly upregulated in Huh7.5 cells by TGF-p, and the ratio of phosphorylated Foxo3a (pFoxo3a) to Foxo3a (pFoxo3a/Foxo3a) was significantly reduced. TGF-β-JNK signaling promoted binding of c-Jun to the Foxo3a promoter region. We constructed Foxo3a promoter l uciferase reporter constructs and showed that mutations introduced into the c-Jun binding element in the Foxo3a promoter abolished the increase in luciferase activity by TGF-β, suggesting that TGF-p-JNK-c-Jun signaling is vital for Foxo3a induction. We also showed that Foxo3a activated Socs3 promoter activity by binding to the Socs3 promoter. To support these findings, TGF-β dose-dependently increased Socs3 expression and inversely suppressed ISG expression. As a result, TGF-β significantly increased HCV replication, and BCAAs suppressed TGFp-JNK-c-Jun signaling, augmented pFoxo3a/Foxo3a expression, and downregulated Socs3 expression. Moreover, BCAAs suppressed TGF-β-SMAD signaling which was activated in patients with advanced liver fibrosis. Finally, TGF-β receptor inhibitor completely blocked TGF-β signaling and decreased HCV replication significantly. Conclusions: TGF-β impairs IFN signaling by activating Socs3-mediated IFN inhibitory signaling via Foxo3a promoter activation by c-Jun binding. BCAAs could be a new therapeutic candidate to augment IFN signaling in HCV replication in patients with advanced CHC.

Disclosures:

Stanley M. Lemon - Advisory Committees or Review Panels: Merck, Santaris, Abbott, Gilead; Consulting: Achillion, Idenix; Grant/Research Support: Merck, Tibotec, Scynexis; Speaking and Teaching: Hoffman LaRoche

Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan

The following people have nothing to disclose: Takayoshi Shirasaki, Masao Honda, Tetsuro Shimakami, Kazuhisa Murai, Takayuki Shiomoto, Hikari Okada, Riuta Takabatake, Seishi Murakami