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A substantial proportion of hepatitis C virus (HCV)-1b-infected patients still do not respond to interferon-based therapy. This study aims to explore a predictive marker for the ultimate virological response of HCV-1b-infected patients treated with pegylated interferon/ribavirin (PEG-IFN/RBV) combination therapy. Nonstructural protein 5A (NS5A) sequences of HCV in the pretreated sera of 45 patients infected with HCV-1b were analyzed. The mean number of mutations in the variable region 3 (V3) plus its upstream flanking region of NS5A (amino acid 2334-2379), referred to as IFN/RBV resistance-determining region (IRRDR), was significantly higher for HCV isolates obtained from patients who later achieved sustained virological response (SVR) by PEG-IFN/RBV than for those in patients undergoing non-SVR. The receiver operating characteristic curve analysis estimated six mutations in IRRDR as the optimal threshold for SVR prediction. Indeed, 16 (76%) of 21 SVR, but only 2 (8%) of 24 non-SVR, had HCV with six or more mutations in IRRDR (IRRDR > 6) (P < 0.0001). All of 18 patients infected with HCV of IRRDR of 6 or greater examined showed a significant (>1 log) reduction or disappearance of serum HCV core antigen titers within 24 hours after initial dose of PEG-IFN/RBV, whereas 10 (37%) of 27 patients with HCV of IRRDR of 5 or less did (P < 0.0001). The positive predictive value of IRRDR of 6 or greater for SVR was 89% (16/18; P ‫؍‬ 0.0007), with its negative predictive value for non-SVR being 81% (22/27; P ‫؍‬ 0.0008). Conclusion: A high degree (>6) of sequence variation in IRRDR would be a useful marker for predicting SVR, whereas a less diverse (<5) IRRDR sequence predicts non-SVR. (HEPATOLOGY 2008;48:38-47.) H epatitis C virus (HCV) infection is the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in industrialized countries. However, HCV infection is curable, and its complications can be prevented by antiviral therapy. 1,2 Currently, the most effective treatment of chronic HCV infection is based on a combination of pegylated interferon (PEG-IFN) and ribavirin (RBV). 3 Even with this treatment regimen, however, sustained virological response (SVR) rates for those infected with the most resistant genotypes, HCV-1a and HCV-1b, still hover at approximately 50%. 3,4 Considering the high cost and the significant side effects associated with this combination therapy, it is worthy to identify patients most likely to benefit from therapy. 5 Predictors of IFN-based therapy can be classified into two categories, pretreatment and on-treatment factors. Pretreatment factors comprise host factors, such as age, sex, obesity, ethanol consumption, hepatic iron overload, fibrosis, immune responses, and coinfection with other viruses, and viral factors, which mainly include viral genotypes and viral load. On-treatment factors are mainly related to the viral kinetics within

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Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells. INTRODUCTIONHepatitis C virus (HCV), a member of the family Flaviviridae, has a single-stranded, positive-sense RNA of about 9?6 kb in length. The virus genome encodes a precursor polyprotein of about 3000 aa, which is cleaved into at least 10 mature viral proteins, such as Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (Reed & Rice, 2000). HCV is known to evade the host-defence mechanisms to establish persistent infection, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (Kiyosawa et al., 1990;Tong et al., 1995). It has been suggested that liver-cell injuries, either apoptotic or necrotic changes, are mediated principally by antiviral immune responses, such as HCV-specific cytotoxic T lymphocytes. However, the possible involvement of viral cytopathic effect (CPE) should also be taken into consideration. Apoptosis involves two major pathways: the Fas-mediated pathway and the mitochondria-mediated pathway (Ashkenazi & Dixit, 1998;Gewies et al., 2000). Fas-mediated apoptosis is conducted through facilitation of caspase-8 activation. Regarding HCV infection, Core was shown to induce Fasmediated and tumour necrosis factor (TNF) receptormediated apoptosis (Ruggieri et al., 1997). On the other hand, a number of apoptosis-inducing signals are concentrated at mitochondria to facilitate the release of cytochrome c from mitochondria, which induces formation of an apoptosome complex that includes apoptosis protease-activating factor-1 (Apaf-1) and procaspase-9 (Deveraux ...

The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) has been implicated in inhibition of antiviral activity of IFN. While previous studies have suggested an interaction between NS5A and the double-stranded RNA-dependent protein kinase (PKR), the possibility still remains that interaction with another molecule(s) is involved in the NS5A-mediated inhibition of IFN. In the present study, we investigated a possible interaction between NS5A and 29,59-oligoadenylate synthetase (2-5AS), another key molecule in antiviral activity. We observed that NS5A physically interacted with 2-5AS in cultured cells, with an N-terminal portion of NS5A [aa 1-148; NS5A(1-148)] and two separate portions of 2-5AS (aa 52-104 and 184-275) being involved in the interaction. Single point mutations at residue 37 of NS5A affected the degree of the interaction with 2-5AS, with a Phe-to-Leu mutation (F37L) augmenting and a Phe-to-Asn mutation (F37N) diminishing it. Virus rescue assay revealed that the full-length NS5A (NS5A-F) and NS5A(1-148), the latter of which contains neither the IFN sensitivity-determining region (ISDR) nor the PKR-binding domain, significantly counteracted the antiviral activity of IFN. Introduction of a F37N mutation into NS5A(1-148) impaired the otherwise more significant IFN-inhibitory activity of NS5A(1-148). It was also found that the F37N mutation was highly disadvantageous for the replication of an HCV RNA replicon. Taken together, our results suggest the possibility that NS5A interacts with 2-5AS and inhibits the antiviral activity of IFN in an ISDR-independent manner.