Hepatitis C virus (HCV) NS2 protein up-regulates HCV IRES-dependent translation and down-regulates NS5B RdRp activity.
ABSTRACT Chronic hepatitis C virus (HCV) infection often leads to liver cancer. The HCV NS2 protein is a hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the function of NS2 protein on HCV replication and translation by using a transient cell-based expression system. Cells co-transfected with pcDNA3.1 (-)-NS2 and the dual-luciferase reporter construct containing the HCV IRES were used to detect the effect of NS2 protein on HCV translation. Cells co-transfected with pcDNA3.1(-)-NS2, pcDNA-NS5B and a reporter plasmid were used to detect the effect of NS2 protein on HCV replication. The results showed that HCV NS2 protein up-regulated HCV IRES-dependent translation in a specific and dose-dependent manner in Huh7 cells but not in HeLa and HepG2 cells, and NS2 protein inhibited NS5B RdRp activity in a dose-independent manner in all three cell lines. These findings may suggest a novel mechanism by which HCV modulates its NS5B replication and IRES-dependent translation and facilitates virus persistence.
- Annual reports in medicinal chemistry 01/2009; 44(1):397-440. DOI:10.1016/S0065-7743(09)04420-0 · 1.36 Impact Factor
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ABSTRACT: Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. In this report, we investigated the function of NS2/3 protease on HCV replication and translation. Cells transfected with plasmids encoding wild-type or mutant NS2/3 and a dual-luciferase reporter construct containing an HCV internal ribosome entry site (IRES) were used to examine the effect of NS2/3 protease on translation of HCV RNA. Cells transfected with plasmids encoding wild-type or mutant NS2/3, pcDNA-NS5B and a reporter plasmid were used to examine the effect of NS2/3 protease on HCV replication. The results showed that both autocleavage processing and the uncleaved form of NS2/3 protease specifically decrease HCV IRES-directed translation, while the uncleaved form of NS2/3 protease decreases HCV NS5B RdRp activity (replication), indicating that autoregulation by NS2/3 protease of HCV replication and translation may play an important role in persistent HCV infection.Archives of Virology 09/2009; 154(9):1465-73. DOI:10.1007/s00705-009-0469-7 · 2.39 Impact Factor
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ABSTRACT: Molecular covariation of highly polymorphic viruses is thought to have crucial effects on viral replication and fitness. This study employs association rule data mining of hepatitis C virus (HCV) sequences to search for specific evolutionary covariation and then tests functional relevance on HCV replication. Data mining is performed between nucleotides in the untranslated regions 5' and 3'UTR, and the amino acid residues in the non-structural proteins NS2, NS3 and NS5B. Results indicate covariance of the 243(rd) nucleotide of the 5'UTR with the 14(th), 41(st), 76(th), 110(th), 211(th) and 212(th) residues of NS2 and with the 71(st), 175(th) and 621(st) residues of NS3. Real-time experiments using an HCV subgenomic system to quantify viral replication confirm replication regulation for each covariant pair between 5'UTR₂₄₃ and NS2-41, -76, -110, -211, and NS3-71, -175. The HCV subgenomic system with/without the NS2 region shows that regulatory effects vanish without NS2, so replicative modulation mediated by HCV 5'UTR₂₄₃ depends on NS2. Strong binding of the NS2 variants to HCV RNA correlates with reduced HCV replication whereas weak binding correlates with restoration of HCV replication efficiency, as determined by RNA-protein immunoprecipitation assay band intensity. The dominant haplotype 5'UTR₂₄₃-NS2-41-76-110-211-NS3-71-175 differs according to the HCV genotype: G-Ile-Ile-Ile-Gly-Ile-Met for genotype 1b and A-Leu-Val-Leu-Ser-Val-Leu for genotypes 1a, 2a and 2b. In conclusion, 5'UTR₂₄₃ co-varies with specific NS2/3 protein amino acid residues, which may have significant structural and functional consequences for HCV replication. This unreported mechanism involving HCV replication possibly can be exploited in the development of advanced anti-HCV medication.PLoS ONE 09/2011; 6(9):e25530. DOI:10.1371/journal.pone.0025530 · 3.23 Impact Factor