Hepatitis C Virus Open Reading Frame Research Articles

Full-genome next-generation sequencing of hepatitis C virus to assess the accuracy of genotyping by the commercial assay LiPA and the prevalence of resistance-associated substitutions in a Belgian cohort.

Although most currently used regimens for Hepatitis C virus (HCV) infections can be initiated without prior knowledge of genotype and subtype, genotyping is still useful to identify patients who might benefit from a personalized treatment due to resistance to direct-acting antivirals (DAA). To assess the utility of full-genome next-generation sequencing (FG-NGS) for HCV genotyping. 138 HCV plasma samples previously genotyped by VERSANT HCV Genotype Assay (LiPA) were subjected to FG-NGS and phylogenetically genotyped Genome Detective. Consensuses were analysed by HCV-GLUE for resistance-associated substitutions (RASs) and their impact on treatment response was investigated. 102/138 (73.9%) samples were sequenced to a genome coverage and depth of >90% of the HCV open reading frame covered by >100 reads/site. Concordant genotype and subtype results were assigned in 97.1% and 79.4% of samples, respectively. FG-NGS resolved the subtype of 13.7% samples that had ambiguous calls by LiPA and identified one dual infection and one recombinant strain. At least one RAS was found for the HCV genes NS3, NS5A, and NS5B in 2.91%, 36.98% and 27.3% samples, respectively. Irrespective of the observed RAS, all patients responded well to DAA treatment, except for HCV1b-infected patients treated with Zepatier (33.3% failure rate (5/15)). While LiPA and FG-NGS showed overall good concordance, FG-NGS improved specificity for subtypes, recombinant and mixed infections. FG-NGS enabled the detection of RAS, but its predictive value for treatment outcome in DAA-naïve patients remains uncertain. With additional refinements, FG-NGS may be the way forward for HCV genotyping.

Comprehensive analysis for viral elements and interleukin-28B polymorphisms in response to pegylated interferon plus ribavirin therapy in hepatitis C virus 1B infection

To comprehensively characterize the contribution of virological factors as well as interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) in determining treatment responses in pegylated-interferon plus ribavirin (Peg-IFN/RBV) therapy for chronic hepatitis C virus (HCV)-1b infection, we undertook a retrospective cohort analysis for the pretreatment dominant complete HCV open reading frame (ORF) amino-acid (aa) sequence study in 103 consecutive HCV-1b Japanese patients. The dominant HCV sequences classified by the response were subjected to systematic sliding-window comparison analysis to characterize response-specific viral sequences, along with IL28B SNP analyses (rs8099917). In each comparison of the patients between with and without rapid viral response (RVR), nonearly viral response (nEVR), sustained virological response (SVR), or relapse, the following regions were extracted as most significantly associated with the different responses respectively: nonstructural protein 5A (NS5A) aa.2224-2248 (P = 1.2E-07); core aa.70 (P = 4E-04); NS5A aa.2340-2382 (P = 7.0E-08); and NS5A aa.2360-2377 (P = 1.1E-05). Those NS5A regions nearly coincided with the interferon (IFN) sensitivity-determining region (NS5A aa.2209-2248) and the IFN/RBV resistance-determining region (NS5A aa.2339-2379). In a multivariate analysis, the IL28B SNP (odds ratio [OR] = 16.8; P = 0.009) and NS5A aa.2340-2382 (OR = 13.8; P = 0.0003) were extracted as the two most-significant independent variables contributing to the final outcome. In Peg-IFN/RBV therapy, polymorphisms in IL28B, NS5A aa.2224-2248, core aa.70, and, most important, NS5A aa.2340-2382 have a tremendous influence on treatment response in association with viral kinetics, resulting in significantly different outcomes in chronic HCV-1b infection.

Hepatitis C virus-induced activation of β-catenin promotes c-Myc expression and a cascade of pro-carcinogenetic events

Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of hepatocellular carcinoma (HCC), although the underlying mechanisms are unclear. The c-Myc oncogene contributes to the genesis of many types of cancers, including HCC, partly via the induction of genetic damage and the inhibition of the cellular response to genotoxic stress. Here, we show a previously undiscovered mechanistic link between HCV infection and enhanced c-Myc expression. c-Myc expression was augmented in non-tumoral liver tissues from HCV-infected individuals with or without HCC and in hepatocyte cell lines harboring an HCV replicon and the infectious HCV strain JFH1. Increased c-Myc expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame, demonstrating a direct role for HCV protein expression in c-Myc induction. Mechanistically, activation of Akt by the HCV non-structural protein NS5A, and the subsequent stabilization of the transcription factor β-catenin, was demonstrated to be responsible for activation of the c-Myc promoter, and for increased c-Myc transcription. β-Catenin-dependent c-Myc expression in this context led to increased production of reactive oxygen species, mitochondrial perturbation, enhanced DNA damage and aberrant cell-cycle arrest. Together, these data provide a novel insight into the mechanisms involved in HCV-associated HCCs, strongly suggesting that c-Myc has a crucial contributory role in this process.

Hepatitis C virus (HCV) protein expression enhances hepatic fibrosis in HCV transgenic mice exposed to a fibrogenic agent

During chronic HCV infection, activation of fibrogenesis appears to be principally related to local inflammation. However, the direct role of hepatic HCV protein expression in fibrogenesis remains unknown. We used transgenic mice expressing the full length HCV open reading frame exposed to a 'second hit' of the fibrogenic agent carbon tetrachloride (CCl(4)). Both acute and chronic liver injuries were induced in these mice by CCl(4) injections. Liver injury, expression of matrix re-modeling genes, reactive oxygen species (ROS), inflammation, hepatocyte proliferation, ductular reaction and hepatic progenitor cells (HPC) expansion were examined. After CCl(4) treatment, HCV transgenic mice exhibited enhanced liver fibrosis, significant changes in matrix re-modeling genes and increased ROS production compared to wild type littermates despite no differences in the degree of local inflammation. This increase was accompanied by a decrease in hepatocyte proliferation, which appeared to be due to delayed hepatocyte entry into the S phase. A prominent ductular reaction and hepatic progenitor cell compartment expansion were observed in transgenic animals. These observations closely mirror those previously made in HCV-infected individuals. Together, these results demonstrate that expression of the HCV proteins in hepatocytes contributes to the development of hepatic fibrosis in the presence of other fibrogenic agents. In the presence of CCl(4), HCV transgenic mice display an intra-hepatic re-organization of several key cellular actors in the fibrogenic process.

Analysis of viral amino acids sequences and the IL28B SNP influencing the development of hepatocellular carcinoma in chronic hepatitis C

The association between hepatitis C virus (HCV) sequences with interleukin 28B (IL28B) single-nucleotide polymorphism (SNP) in the development of hepatocellular carcinoma (HCC) has not been well clarified. Complete HCV open-reading frame sequences were determined in 20 patients developing HCC and 23 non-HCC patients with HCV-1b infection in two distant time points. An additional 230 patients were studied cross-sectionally for core and NS5A sequences with HCC development. Among them, 98 patients with available samples were investigated for changes in viral core sequences over time. Finally, IL28B SNPs and HCC development were investigated in 228 patients. During observation period (HCC for 10.8years, and non-HCC for 11.1years), changes in core a.a. 70 and three amino acid positions in NS5A were characteristics of the patients developing HCC. In 230 patients, Q (glutamine) or H (histidine) to R (arginine) ratio at core a.a. 70 was significantly higher in the HCC group (HCC group 43:22 vs. non-HCC group 66:99, p=0.001). A change in core R70Q was observed over time in 11 patients associated with a decrease in platelets (p=0.005) and albumin (p=0.005), while a Q70R change was observed in 4 patients without associated changes in platelets (nonsignificant) and albumin (nonsignificant). IL28B SNP showed significant correlation with the core a.a. 70 residue. There was no evident link between IL28B SNPs and the occurrence of HCC. Hepatitis C virus core a.a. 70 residue is associated with liver disease progression and is independent factor for HCC development in genotype-1b infection. IL28B SNPs are related to core a.a. 70 residue, but not to HCC. The functional relevance of core a.a. 70 residue in hepatitis C pathogenesis should be further investigated.

Analysis of the complete open reading frame of hepatitis C virus in genotype 2a infection reveals critical sites influencing the response to peginterferon and ribavirin therapy

A proportion of patients infected with genotype 2a hepatitis C virus (HCV) cannot achieve a sustained virological response (SVR) to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV) but the reason remains unclear. The present study aimed to clarify the possible correlation between viral sequence variations and final outcome. The pretreatment complete open reading frame (ORF) sequences of genotype 2a HCV were determined by direct sequencing for two independent groups of patients (43 patients as test; group 1 and 35 as validation; group 2), and the correlation with the final outcome was explored. Patients with SVR (n=58) and with non-SVR (n=20) differed significantly in pretreatment HCV RNA level (p=0.002), fibrosis score (p=0.047), and cumulative RBV dosage (p=0.003). By comparison of all amino acid positions in the complete HCV ORFs, threonine at amino acid (aa) 110 in the core region was remarkably frequent in SVR (p=0.01 for group 1, p=0.004 for group 2, and p=5E-05 for combined). A sliding window analysis revealed that the total number of amino acid variations within the NS5A aa 2258-2306 region were significantly high in SVR compared to non-SVR patients (p=0.01 for group 1, p=0.006 for group 2, and p=0.0006 for combined). Multivariate analyses revealed that core aa 110 (p=0.02), NS5A aa 2258-2306 (p=0.03), and cumulative RBV dosage (p=0.02) were identified as independent variables associated with the final outcome. The outcome of PEG-IFN/RBV therapy is significantly influenced by variation in the core and NS5A regions in genotype 2a HCV infection.

Downregulation of Gadd45β Expression by Hepatitis C Virus Leads to Defective Cell Cycle Arrest

Members of the Gadd45 family play central roles in the cellular response to genotoxic stress and have been implicated in several human cancers, including hepatocellular carcinomas. Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of primary hepatocellular tumors, although the underlying mechanisms are unclear. Here, we show a novel link between diminished Gadd45beta expression and HCV infection. Inhibited Gadd45beta expression was observed in both nontumoral and tumoral tissues from infected individuals, and in cell lines harboring a HCV replicon and the infectious HCV strain JFH1. Decreased Gadd45beta expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame. Mechanistically, hypermethylation of the Gadd45beta promoter in the presence of HCV is responsible for this defect. Diminished Gadd45beta expression leads to aberrant cell cycle arrest and diminished DNA excision repair. Together, these results provide a novel insight into the mechanisms involved in HCV-associated hepatocellular carcinomas, showing that reduced Gadd45beta expression may play a contributory role to this process, and providing evidence that HCV may interfere with epigenetic gene expression by altering promoter methylation.

874 EPIGENETIC MODULATION OF TUMOUR SUPPRESSOR GADD45 BETA EXPRESSION BY HCV LEADS TO DEFECTIVE CELL CYCLE ARREST AND MAY PLAY A ROLE IN LIVER CARCINOGENESIS

Background and Aims: Chronic hepatitis C virus (HCV) infection is a major risk factor of hepatocellular carcinoma (HCC). Sustained oxidative stress and elevated DNA damage are considered major contributors to HCC. Gadd45b is a major mediator of the cellular response to DNA damage. Our goal was to study the effect of chronic HCV infection on Gadd45b expression. Methods: Gadd45b expression were analyzed by western blotting or quantitative RT-PCR in: (a) paired non-tumoral and tumoral human liver samples from uninfected patients, and patients chronically infected with hepatitis B virus (HBV) or HCV; (b) Huh7 cell cultures harboring the HCV genotype 1b replicon and the JFH1 infectious strain; (c) liver tissues from 18 month old wild-type (wt) and transgenic mice expressing the full-length HCV open reading frame (FL-N/35). Primary hepatocytes were isolated from 7 month old mice. Promoter hypermethylation was assessed by the effect of 5-azacytidine (a DNA methyltransferase inhibitor) on Gadd45b mRNA expression. Cell cycle arrest and mitotic indices were assessed by the presence of phospho-histone H3 after UV-C irradiation. Results: Gadd45b expression was abrogated in tumoral tissue from HBV-positive or -negative patients, whereas it was found at normal levels in non-tumoral tissues. In contrast, in HCV-infected individuals, Gadd45b was absent from both tumoral and nontumoral tissues. Gadd45b expression was also reduced in Huh7 cells exhibiting HCVRNA replication (although these cells originate from a liver tumor). A 2-fold decrease in levels of Gadd45b mRNA was observed in livers of FL-N/35 mice compared to controls, whereas amounts of related Gadd family members remained unaffected. Activation of the Gadd45b promoter was inhibited in the presence of HCV, with cells isolated from FL-N/35 mice demonstrating a 2-fold reduction in promoter activity compared to wt controls. Reduced murine Gadd45b expression was associated with decreased cell cycle arrest in response to DNA damage, and both Gadd45b expression and cell cycle arrest were restored to wt levels by treatment with 5-azacytidine. Conclusions: These data demonstrate that HCV protein expression is associated with inhibition of Gadd45b expression through hypermethylation of its promoter, adversely altering the DNA damage response and potentially promoting liver carcinogenesis.

Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor kappaB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation.

Novel Chimeric Genotype 1b/2a Hepatitis C Virus Suitable for High-Throughput Screening

A major obstacle in hepatitis C virus (HCV) research has been the lack of a permissive cell culture system that produces infectious viral particles. Significant breakthroughs have been achieved lately in establishing such culture systems. Yet to date, there are no reports of the applications of any of these systems in HCV drug screening. Here, we report the generation of two monocistronic, chimeric genotype 1 full-length HCV genome molecules. These molecules, C33J-Y835C-UBI and C33J-Y835C-FMDV2A, both contain the structural protein region from genotype 1 (subtype 1b, Con1) and the remaining region from the genotype 2a (JFH1) clone. Both contain the humanized Renilla luciferase reporter gene which is separated from the rest of the HCV open reading frame by two different cleavage sites. The viral RNAs replicated efficiently in transfected cells. Viral particles produced were infectious in naïve Huh7.5 cells, and the infectivity could be blocked by monoclonal antibody against a putative HCV entry cofactor, CD81. A pilot high-throughput screen of 900 unknown compounds was executed by both the genotype 2a subgenomic replicon system and the infectious system. Thirty-one compounds were identified as hits by both systems, whereas 78 compounds were identified as hits only for the infectious system, suggesting that the infectious system is capable of identifying inhibitors targeting the viral structural proteins and steps involving them in the viral life cycle. The infectious HCV system developed here provides a useful and versatile tool which should greatly facilitate the identification of HCV inhibitors currently not identified by the subgenomic replicon system.

Hepatitis C virus RNA: Dinucleotide frequencies and cleavage by RNase L

Ribonuclease L (RNase L) is an antiviral endoribonuclease that cleaves hepatitis C virus (HCV) RNA at single-stranded UA and UU dinucleotides throughout the open reading frame (ORF). To determine whether RNase L exerts evolutionary pressure on HCV we examined the frequencies of UA and UU dinucleotides in 162 RNA sequences from the Los Alamos National Labs HCV Database ( http://hcv.lanl.gov). Considering the base composition of the HCV ORFs, both UA and UU dinucleotides were less frequent than predicted in each of 162 HCV RNAs. UA dinucleotides were significantly less frequent than predicted at each of the three codon positions while UU dinucleotides were less frequent than predicted predominantly at the wobble position of codons. UA and UU dinucleotides were among the least abundant dinucleotides in HCV RNA ORFs. Furthermore, HCV genotype 1 RNAs have a lower frequency of UA and UU dinucleotides than genotype 2 and 3 RNAs, perhaps contributing to increased resistance of HCV genotype 1 infections to interferon therapy. In vitro, RNase L cleaved both HCV genotype 1 and 2 RNAs efficiently. Thus, RNase L can cleave HCV RNAs efficiently and variably reduced frequencies of UA and UU dinucleotides in HCV RNA ORFs are consistent with the selective pressure of RNase L.

Generation of inducible hepatitis C virus transgenic mouse lines

Hepatitis C virus (HCV) is the causative agent of most cases of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) affecting more than 170 million people world-wide. Progress in elucidating the nature of HCV and the development of new therapeutic strategies is hampered fundamentally by the absence of adequate small animal models simulating natural HCV infection. The creation of conditional mouse lines with the tetracycline-controlled gene expression system holds new perspectives for simulation of wild-type HCV infection in a small animal model. Transgenic mice were established with tetracycline-inducible coexpression of HCV core or HCV open reading frame (ORF) and luciferase. In long-term induction experiments, mice were examined for immunopathological changes after expression of HCV proteins. Inducible and liver-specific expression of transgenes was detected by Western blot, immunoprecipitation, luciferase assay and in vivo imaging of bioluminescence of luciferase in genetically modified mice. Ectopic expression levels were determined quantitatively in the liver, kidney, heart and spleen of mice in the induced and non-induced state. During long-term induction an elevation of aminotransaminases (ALT) was observed only in HCV core/ORF-expressing mice, but HCV-specific immune response was not confirmed by in vitro immunological assays. The histology of liver sections provided evidence of steatosis, which was correlated with an inflammatory response. The inducible HCV-transgenic mouse lines provide further evidence of liver pathogenesis in the presence of inflammation during liver-specific expression of HCV proteins and offer new insights into the effects of temporally and spatially controlled protein expression of HCV.

Hepatitis C virus protein expression causes calcium-mediated mitochondrial bioenergetic dysfunction and nitro-oxidative stress

Hepatitis C virus (HCV) infection induces a state of oxidative stress that is more pronounced than that in many other inflammatory diseases. In this study we used well-characterized cell lines inducibly expressing the entire HCV open-reading frame to investigate the impact of viral protein expression on cell bioenergetics. It was shown that HCV protein expression has a profound effect on cell oxidative metabolism, with specific inhibition of complex I activity, depression of mitochondrial membrane potential and oxidative phosphorylation coupling efficiency, increased production of reactive oxygen and nitrogen species, as well as loss of the Pasteur effect. Importantly, all these effects were causally related to mitochondrial calcium overload, as inhibition of mitochondrial calcium uptake completely reversed the observed bioenergetic alterations. Expression of HCV proteins causes deregulation of mitochondrial calcium homeostasis. This event occurs upstream of further mitochondrial dysfunction, leading to alterations in the bioenergetic balance and nitro-oxidative stress. These observations provide new insights into the pathogenesis of hepatitis C and may offer new opportunities for therapeutic intervention.

Pretreatment Sequence Diversity Differences in the Full-Length Hepatitis C Virus Open Reading Frame Correlate with Early Response to Therapy

Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.

Expression of Toll‐like receptors in chronic hepatitis C virus infection

Toll-like receptors (TLRs) are involved in innate immunity. Certain viruses interact with TLRs and mediate antiviral effects as well as immune responses. The aim of this study was to investigate the effect of TLRs on pathogenesis in hepatitis C virus (HCV)-infected patients. Peripheral blood mononuclear cells (PBMC) and CD14+ (monocytes) or CD14- cells from 25 patients with chronic liver disease and 15 healthy subjects were studied for expression of TLRs 2-9 and cytokines of extracted RNA using real-time PCR. Then TLR expression was examined in HepG2 cells transfected with entire or parts (core-NS3, NS3-NS5B) of the HCV open reading frame. TLR expression was calculated as the relative mRNA levels. Expression of TLRs 4, 7 and 8 in CD14+ cells of PBMC was increased in patients. Levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p35 for PBMC were also increased in patients. When PBMC were incubated with HCV core protein, enhancement of TLR2 expression and suppression of TLR4 and TLR7 were noted in patients. Similar alteration of TLRs expression was observed in controls. Among HepG2 transfectants, only TLR3 expression was changed; it was suppressed in entire gene transfectant and enhanced in core-NS3 transfectant. Expression of some proteins related to the TLR signaling pathway was suppressed in the entire gene transfectant. The results suggest a correlation between expression levels of TLRs and cytokines, and chronic HCV infection. TLR3 recognizes double-stranded RNA and induces type 1 interferon synthesis. Collectively, suppressed expression of TLR3 in cells transfected with entire HCV may be responsible for continuous HCV infection, although a part of the HCV gene enhances its expression.

Generation of inducible hepatitis C virus transgenic mouse lines

Background and Aims: Hepatitis C virus (HCV) is the causative agent for most cases of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) affecting more than 170 million people world-wide. Progress in elucidating the nature of HCV and the development of new therapeutic strategies is fundamentally hampered by the absence of adequate small animal models simulating natural HCV infection. The creation of conditional mouse lines with the tetracycline-controlled gene expression system holds new perspectives for simulation of wild-type HCV infection in a small animal model. Methods: Transgenic mice were established with tetracycline-inducible coexpression of HCV core or HCV ORF (open reading frame) and luciferase. In long-term induction experiments, mice were examined for immunopathology after expression of HCV proteins. Results: Inducible and liver-specific expression of transgenes was detected by Western blot, immunoprecipitation, luciferase assay and in vivo imaging of bioluminescence of luciferase in genetically modified mice. Ectopic expression levels were determined quantitatively in the liver, kidney, heart and spleen of mice in induced and non-induced state. During long-term induction an elevation of aminotransaminases (ALT) was observed only in HCV core/ORF-expressing mice, but no HCV-specific immune response was confirmed by in vitro immunological assays. The histology of liver sections gained evidence for steatosis, which was in correlation with an inflammatory response. Conclusions: The conditional HCV-transgenic mouse lines give further evidence of liver pathogenesis in the presence of inflammation during liver-specific expression of HCV proteins and offer new insights into the effects of temporally and spatially controlled HCV protein expression.

A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

BackgroundHepatitis C virus (HCV) is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription-polymerase chain reaction (RT-PCR) of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile.ResultsRT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF) from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon) for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two sets of sequencing primers were optimized for each genotype 1a and 1b. Viral consensus sequences were determined by directly sequencing the amplicons. HCV ORFs from 72 patients have been sequenced using these primers. Sequencing errors were negligible because sequencing depth was over 4-fold and both strands were sequenced. Primer bias was controlled and monitored through careful primer design and control experiments.ConclusionOptimized RT-PCR and sequencing conditions are useful for rapid and reliable amplification and sequencing of HCV genotype 1a and 1b ORFs.

Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice.

To assess the effects of constitutive hepatitis C virus (HCV) gene expression on liver, transgenic mice carrying the entire HCV open reading frame inserted in the alpha1 antitrypsin (A1AT) gene were generated. Expression of A1AT/HCV mRNA was found to be mainly limited to perivascular areas of the liver as indicated by in situ hybridization analysis. HCV core protein was detected in Western blots of liver extracts, whereas the expression of E2, NS3 and NS5 proteins was revealed by immunostaining of liver samples using HCV-specific antisera. Histological analysis of HCV transgenic mice showed that these animals develop extensive steatosis, but very little necrosis of liver tissue. Moreover, a consistent T cell infiltrate and a slight hepatocyte proliferation were observed. Phenotypic analysis of cells infiltrating the liver indicated that recruitment and/or expansion of residing CD8(+), NK, NKT and gammadelta T cells occurred in transgenic animals. Among these cells, a large fraction of CD8(+) T lymphocytes released mainly IL-10 and, to a lesser extent, IFN-gamma upon mitogenic stimulation in vitro. Furthermore, both intrahepatic lymphocytes and splenocytes did not produce cytokines in response to HCV antigens. Thus, these data indicate that constitutive expression of HCV proteins may be responsible for intrahepatic lymphocyte recruitment in absence of viral antigen recognition. This response is likely to be driven by virus-induced cellular factors and may play a significant role in the immunopathology of chronic HCV infection and liver disease.

Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame

Although hepatitis C virus (HCV) is a causative agent of liver diseases, its mechanism of pathogenesis is still unclear, mainly because of the lack of adequate cell culture systems to support HCV infection and replication. In this report, we describe development and characterization of human hepatoma cell lines constitutively expressing entire (Hep394) or parts (Hep352, Hep3294) of the HCV open reading frame (ORF). The viral and cellular proteolytic machinery involved in the viral precursor processing was consistently functional, and processed HCV proteins were synthesized in these established cell lines. By using a cDNA microarray analysis coupled with semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), we identified 12 genes up-regulated and 4 genes down-regulated in Hep394 cells. With regard to genes related to cell growth regulation, we found up-regulation of forkhead transcription factor FREAC-1, poly (A) binding protein PABP2, and Ras suppressor Rsu-1. Another category of changes in gene expression includes MHC antigens, which play an important role in the T-cell-mediated immune reaction in the liver. In conclusion, functional genomic approaches comparing expression among the different cell lines expressing parts of the HCV genome may promote our understanding of the molecular basis of pathogenicity of HCV infection. (H EPATOLOGY 2002;36:1431-1438.)

Expression of hepatitis C virus proteins inhibits signal transduction through the Jak-STAT pathway.

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. Alpha interferon (IFN-alpha) therapy of chronic hepatitis C leads to a sustained response in 10 to 20% of patients only. The mechanisms of viral persistence and the pathogenesis of hepatitis C are poorly understood. We established continuous human cell lines, allowing the tightly regulated expression of the entire HCV open reading frame under the control of a tetracycline-responsive promoter. Using this in vitro system, we analyzed the effect of HCV proteins on IFN-induced intracellular signaling. Expression of HCV proteins in these cells strongly inhibited IFN-alpha-induced signal transduction through the Jak-STAT pathway. Inhibition occurred downstream of STAT tyrosine phosphorylation. Inhibition of the Jak-STAT pathway was not restricted to IFN-alpha-induced signaling but was observed in leukemia inhibitory factor-induced signaling through Stat3 as well. By contrast, tumor necrosis factor alpha-induced activation of the transcription factor NF-kappaB was not affected. Interference of HCV with IFN-alpha-induced signaling through the Jak-STAT pathway could contribute to the resistance to IFN-alpha therapy observed in the majority of patients and may represent a general escape strategy of HCV contributing to viral persistence and pathogenesis of chronic liver disease.