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HCV Targets TLR7 Receptor Expression By Reducing TLR7 mRNA Stability inHCV Replicating CellsSerena Chang, Gyongyi Szabo

The increasing prevalence and ability to evade the host innate immune system aids theHepatitis C Virus (HCV), a single-stranded RNA virus, to develop into a chronic andpotentially lethal infection. Members of the Toll-like receptor (TLR) family were implicatedin having a therapeutic role in controlling HCV. Endosomal TLRs, TLR7 or -8 activated bysingle-stranded RNA, produce Type 1 interferons via phosphorylation of interferon regulatoryfactors (IRFs). Preclinical and clinical studies demonstrated a decrease in HCV viral replicationwith TLR7 agonist administration. The purpose of this study was to investigate whether theTLR7 receptor was a target of HCV infection. We used human hepatoma cell lines expressingHCV full-length (FL) and sub-genomic (BB7) genotype 1b, and hepatoma cells infected withthe HCV infectious clone (JFH-1) of genotype 2, to evaluate TLR7 mRNA and protein levels.Base line levels of TLR7 mRNA by QRT-PCR, in HCV cells showed a 70% reduction comparedto non-HCV cells. TLR7 protein levels by flow cytometry showed a 50, 45, and 24%reduction in JFH-1, FL, and BB7 cells. Micro-array analysis of HCV infected patient liversalso demonstrated a decrease in TLR7 expression compared to non-HCV infected controls.Next, we investigated the effect of HCV infection on the reduction of TLR7 by stimulatingHCV cells with IFNα and examined TLR7 expression. IFNα reduced HCV mRNA levels byover 70% in HCV cells compared to un-stimulated controls which correlated with a significantincrease in TLR7 mRNA and protein expression. With TLR7 expression linked to HCVinfection, we explored the functional effects on the TLR7 downstream pathway by measuringlevels of IRF7 nuclear translocation upon TLR7/8 ligand R848 stimulation. In non-HCVcells we observed a significant 2-6 fold increase as opposed to a 10% decrease in HCVreplicating cells in IRF7 nuclear translocation after stimulation with R848. With the loss ofTLR7 expression and pathway function due to HCV infection, we examined mRNA degrada-tion rates as a possible molecular mechanism using RNA transcriptional inhibitors. In HCVcells, the half life of TLR7 was within 2 hours of mRNA inhibition compared to non-HCVcells with a half life of 4 hours. This increased degradation rate in HCV cells is specific toTLR7 as both TLR4 and TLR5 had equivalent or increased half lives compared to non-HCVcells. These results demonstrate an attenuation of TLR7 expression and function in HCVinfection of hepatocyte cell lines due to increased degradation of TLR7 mRNA. ReducedTLR7 expression may represent an additional mechanism by which HCV prevents sufficientinnate immune responses for viral elimination.

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Mutations in Epitopes Restricted By Protective HLA A3 and HLA DRB1 0401Alleles Are Associated with the Development of Persistent Hepatitis CInfectionDanijela Petrovic, Eugene Dempsey, Karen Fitzmaurice, Susan M. McKiernan, AideenLong, Dermot P. Kelleher

A study of an Irish cohort of females infected with HCV 1b from a single source demonstratedthat the HLA-A3 and HLA DRB1 0401 alleles were significantly associated with viral clearance.HLA-A3 allele was expressed in 39.5 % while DRB1 0401 was present in 29.1 % of patientsclearing the virus as compared to 19.1 % with viral persistence for A3 and 15.6% for DRB10401. Hence, in order to identify mechanisms of viral clearance in this cohort, it is criticalto identify epitopes from HCV 1b restricted by these protective alleles. The aim of this studywas to utilise a bio-informatics approach to identify epitopes and to compare to viralsequences obtained from HLA-A3 and DRB1 0401 positive and negative patients. Weconstructed a detailed model utilising HLA Class I and II epitope predicting databases. ViralRNA was extracted from patients' serum samples, amplified and sequenced. CLC Bio andBioEdit software packages were used to perform analysis of viral sequence data. This involvedin silico protein translation, alignment with the donor sequence and epitope mapping.Predicted epitopes were aligned with sequences obtained from 15 HLA-A3 and 7 DRB10401 non-resolvers and controls (17 non-HLA-A3, 17 non-DRB1 0401 all non-resolvers).Significant amino acid substitutions were observed in 2 epitopes for HLA-A3 and one forDRB1 0401. The remaining predicted epitopes were either conserved in both groups orlocated in the parts of viral genome not yet sequenced. In the HLA-A3 group of non-resolvers, 8 out of 15 (53.33%) had a substitution of lysine (K) for arginine (R) in theKLTPPHSAK epitope compared with the non HLA-A3 group with one out of 17 patientshaving the substitution (5.88%)(p= 0.004). 77.77% of this HLA-A3 group had a K to Rsubstitution in the TVYHGAGTK epitope, which was not present in the non-HLA-A3 group(p=0.0001). This mutation occurred singly or in combination with substitution of threonine(T) to alanine (A) (62.5%). Again, this double mutation was not observed in the controlgroup (p=0.002). Substitution of T to A was present in 5 HLA-A3 positive patients out of8 available sequences (62.5%) and in just one of 13 available sequences in the control group(7.69%) (p=0.014) In the DRB1 0401 group, 42.9% expressed a valine (V) to isoleucine (I)substitution in the HHNMVYATTSRSASQ epitope. None of the non-DRB1 0401 patientshad this change (p=0.017). This bioinformatic approach has identified novel candidate HCV1b epitopes. Mutations observed within these HLA restricted epitopes may contribute toviral immune escape and represent a mechanism whereby HCV can establish persistentinfection and chronic disease.

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Impaired Hepatic Lipid Metabolism By Hepatitis C Virus in Mice andHumans: Implications for Modulation of Transcriptional ProcessesMichael Kremer, Arash Nickkholgh, Richard J. Milton, Michael D. Wheeler, Richard A.Rippe, Peter Schemmer, Ian N. Hines

A growing body of evidence suggests a direct impact of Hepatitis C virus (HCV) on hepaticfat accumulation. The purpose of this study was to determine the activity of the transcriptionfactors PPARγ and SREBP1 and their downstream effector enzymes in the presence of HCV.Wild type (Wt) mice and transgene mice expressing the HCV proteins (HCVtg) received

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choline deficient diet for 16 weeks to supplement hepatocellular lipid uptake. HCVtg micepresented with significant increases in hepatic triglyceride content (27.4±17.69mg/g vs.132±55.31mg/g for wt vs. HCVtg fed CDD; p<0.05) which correlated with impaired inductionof components of hepatic fatty acid metabolism including fatty acid binding protein, acylCoA oxidase and very long chain acyl CoA synthetase assessed by real-time PCR. To furtherinvestigate this as a potential mechanism present within HCV infected human livers, humanliver tissue was obtained during liver transplantation from explanted livers due to HCV-associated cirrhosis. Samples from ethanol-induced liver cirrhosis served as internal control.Similar to the mechanisms identified in mouse, hepatic HCV infection in humans alsoimpairs the expression of fatty acid binding protein, acyl CoA oxidase and very long chainacyl CoA synthetase 2 gene expression in conjunction with significantly increased PPARγnuclear localization as assessed by western blot and changes in SREBP DNA binding com-plexes as assessed by electrophoretic mobility shift assay. In summary, the presence of HCVin a transgene mouse model and in humans with end stage liver cirrhosis results in impairedhepatic lipid metabolism. We present a potential mechanism that explains the initial increasein hepatic lipid accumulation after HCV infection. Modulation of this pathway might proveto be useful in a clinical setting of Hepatitis C infection with amelioration of the onset andseverity of HCV-associated hepatosteatosis, a pathology which likely contributes to increaseddevelopment of hepatic fibrosis. This work was supported by NIAAA grants AA014243,K01AA016563, and F32-AA015005.

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A Dramatic Alteration in the Cholesterol Metabolism in An HCV Infected-Liver: the Implications for HCV Infection and TreatmentMakoto Nakamuta, Tatsuya Fujino, Ryoko Yada, Masayoshi Yada, Tsuyoshi Yoshimoto,Ryosuke Takemoto, Kunitaka Fukuizumi, Naohiko Harada, Nobito Higuchi, Masaki Kato,Kazuhiro Kotoh, Munechika Enjoji

Background/Aims: Recently, a close relationship between HCV infection and the cholesterolmetabolism has been reported: namely, HCV infects hepatocytes via LDL receptor (LDLR);The LDL levels inversely are correlated with the HCV levels; statin suppresses HCV replicationIn Vitro; The LDL levels affect the outcome of IFN treatment. In human HCV infection,however, little is known about the cholesterol metabolism in hepatocytes. We thereforeinvestigated the cholesterol metabolism related-gene expression in the HCV-infected liver.Methods: We performed real-time PCR using samples obtained from an HCV-infected liver(n=70, genotype 1b=44, genotype 2=26) and also from a normal liver (n=10). The targetgenes for a real-time PCR analysis were as follows: LDLR, HMG-CoA reductase (HMG-CoAR), MTP, SREBP-2, and LXRα. Results: The expression of LDLR dramatically decreasedby 98% in the HCV-infected liver in comparison to the normal liver. In contrast, theexpression of HMG-CoA reductase was enhanced 2-fold in the HCV-infected liver. Theexpression of SREBP-2, which positively regulates the expression of LDLR and HMG-CoARsynchronously, decreased by 70%. The expression of MTP and LXRα increased 3-fold and1.5-fold, respectively. No major difference in the gene expression patterns were observedbetween HCV genotype 1b and 2. Conclusion: These data indicate cholesterol accumulationin the HCV-infected liver. Cholesterol accumulation generally leads a down-regulation ofHMG-CoAR; however, such accumulation was was observed to be inversely up-regulatedin the infected liver, suggesting that HCV might directly affect cholesterol synthesis. Thesedata also suggest that cholesterol metabolism modulators, such as statin and ezetimibe,might therefore beneficially affect HCV therapy. We are now investigating the relationshipbetween the effects of IFN+ribavirin therapy and the gene expression.

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Steatosis in Chronic Hepatitis C: Changes in Hepatic Fatty Acid CompositionBianca M. Arendt, Ellie Aghdassi, Saira Mohammed, David W. Ma, Jenny Heathcote,Johane P. Allard

Hepatic steatosis is a cofactor of disease in chronic Hepatitis C (HCV), which acceleratesdisease progression and lowers the response rate to antiviral therapy. Hepatic fatty acid (FA)composition, especially n-6 and n-3 polyunsaturated FA (PUFA), and oxidative stress mightplay an important role in the development of hepatic steatosis. Aim was to compare hepaticFA composition and oxidative stress in patients with HCV and steatosis to those withoutsteatosis. METHODS: This is a cross-sectional study. Liver biopsies were obtained from 38patients with HCV (10 with steatosis ≥5% of hepatocytes, 28 without steatosis). HepaticFA composition in % of total lipids was measured by gas chromatography. Lipid peroxidationin the liver, antioxidant potential, and TNF-α in liver and plasma (testkits), and plasmaantioxidant vitamins (HPLC) were determined. Patients completed 3-day food records and7-day activity logs, and anthropometric data and blood biochemistry (liver enzymes, fastingglucose, insulin, HbA1c, lipid profile) were assessed. Wilcoxon Signed-Rank or Chi-squaretest we applied. RESULTS: Patients with steatosis had lower concentrations of total PUFA,n-3 PUFA, and eicosapentaenoic acid in hepatic total lipids than those without steatosis. n-6/n-3 ratio and essential FA (linoleic and linolenic acid) were not altered, but the ratios ofactive metabolites to their essential FA precursors were reduced in steatosis. Plasma vitaminC was also reduced (no steatosis vs. steatosis: 1.1±0.1 vs. 0.7±0.1mg/dL; p=0.016), whereasother markers of oxidative stress were not altered. Patients with steatosis had a higher bodymass index (24.1±0.6 vs. 29.4±1.9 kg/m2; p=0.006) and waist-to-hip ratio (0.89±0.02 vs.0.99±0.02; p=0.012), but dietary intake, physical activity, or blood biochemistry were notdifferent. CONCLUSIONS: Patients with HCV and steatosis have reduced n-3 PUFA inhepatic total lipids compared to patients without steatosis. Lower metabolite/precursor ratiosfor n-6 and n-3 PUFA suggest changes in hepatic FA metabolism. In contrast, oxidativestress does not seem to play a predominant role for steatosis in HCV.Fatty acid composition in hepatic total lipids of patients with chronic Hepatitis C