Molecular Mechanisms of Interferon Resistance Mediated by NS3, NS4A and NS4B Genes of Hepatitis C Viruses in Chronic Hepatitis C Patients Treated with Peginterferon-α and Ribavirin

Abstract

Several HCV genotypes circulating in Thailand has been reported, including HCV-1a, -1b, -3a, -3b and various subtypes of HCV-6. Standard regimen for the treatment of hepatitis C is based on the combination of pegylated inferferon-α (IFN-α) and ribavirin (RBV). The NS3 is a multifunctional protein, with a serine protease and helicase domains. The NS4A protein functions as a cofactor for the NS3 serine protease. The NS3-NS4A protease is responsible for cleavage of HCV polyprotein into several functional proteins while helicase is essential for HCV RNA replication and also plays a role in viral particle assembly. Several studies suggest that NS3-NS4A protease could be one of the factors that HCV uses to interfere with the host antiviral response. In addition, NS4B has been found to be responsible for the formation of the membranous web, which is the platform for viral replication. We hypothesized that amino acid variations in the NS3, NS4A and NS4B proteins may affect the functions of those proteins, and therefore interfere with the HCV replication. The present study aimed to assess the effects of HCV amino acid variations in NS3, NS4A and NS4B of HCV on the response to Peg-IFN and RBV treatment and also to investigate the inhibitory effects of NS3-NS4A proteins on the IFN-α signaling pathway by HCV-1a, -1b, -3a, -3b, and -6f. One hundred and thirty four patients infected with HCV-1a, -1b, -3a, -3b and -6f strains that showed different clinical outcomes, the responders and treatment failure group, at before treatment and at week 4 of treatment were evaluated.

The full-length NS3, NS4A and NS4B genes of HCV obtained at before and at week 4 of treatment were amplified by reverse transcription polymerase chain reaction (RT-PCR) and then the PCR products were purified and sequenced. Amino acid sequences of NS3, NS4A and NS4B were analyzed in comparison with reference sequences of corresponding genotypes. The data revealed that amino acid variations within the full-length, protease and helicase domains of NS3 but not in the NS4A and NS4B of HCV-1a from responders at before treatment were significantly higher than those from treatment failure group as compared with reference sequences. Similar results were observed in the full-length and helicase domain but not in protease domain of HCV-1b. However, the number of amino acid variations in NS3, NS4A and NS4B of HCV-3a, -3b, and -6f from responders and treatment failure group were not significantly different. Furthermore, comparison of amino acid variations between HCV obtained at before treatment and these obtained at week 4 of treatment also revealed that the mean number of amino acid variations in full-length, protease and helicase domains of NS3 of HCV-3a and -3b but not HCV-1a, -1b, and -6f from responders were also significantly higher than those from treatment failure group.

Study on the inhibitory effects of NS3-NS4A and NS4B proteins on IFN-α- induced interferon signaling pathway was performed by using NS3-NS4A and NS4B over-expression system. The NS3-NS4A or NS4B of HCV-1a, -1b, -3a, -3b, or -6f were expressed by co-transfecting a plasmids encoding pCAGGS-NS3-NS4A or pCAGGS-NS4B together with pISRE-luc into Huh7.5.1 cells in the presence of IFN-α. The JFH1-infected Huh7.5.1 cells and empty vector transfected Huh7.5.1 cells were used as a positive and negative controls, respectively. The effects of NS3-NS4A or NS4B proteins on the IFN signaling were assessed. Whole cell lysates were harvested to measure relative luciferase activity of pISRE signaling and phosphorylations of STAT1 and STAT2 were determined by western blot analysis. The levels of mRNA specific for PKR, OAS, and MxA were quantified by real-time PCR in order to measure the expression levels of interferon-stimulated genes (ISGs). The results showed that NS3-NS4A of HCV-1a and -1b but not HCV-3a, -3b, and -6f significantly inhibited ISRE signaling by reducing the phosphorylation of STAT2, and mRNA levels specific for PKR, OAS and MxA.

In summary, the increase of amino acid variations within the NS3 protein but not in NS4A and NS4B were associated with the response to Peg-IFN and RBV treatment in Thai patients. Our studies also provided the new insight into the mechanisms by which HCV NS3-NS4A protein interfered with the interferon- signaling pathway by inhibiting the expression of ISGs through the reduction of STAT2 phosphorylation and mRNA levels of PKR, OAS and MxA.