Abstract
We have previously shown that physio/pathological levels of hydrogen peroxide (H2O2) stimulate translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) element in tissue-cultured cells. Here, using in vitro translation, we further show that H2O2 upregulates HCV IRES-dependent mRNA translation and correlates with an increase in intracellular oxidant level. Using Western blotting, immunocytochemistry, microscopy and affinity pulldown, we show that H2O2 stimulates HCV IRES-dependent translation and correlates with nuclear–cytoplasmic shuttling of the La autoantigen, resulting in enhanced binding of cytoplasmic La to HCV IRES RNA. The role of the La protein in H2O2-stimulated IRES-dependent translation is further confirmed by the ability of an anti-La antibody to suppress H2O2-activated IRES-dependent translation in vitro. This is further supported by the ability of an ectopically expressed dominant, negative La mutant protein to suppress H2O2-inducible IRES-mediated translation in Huh7 cells, transiently transfected with a bicistronic reporter and in a sub-genomic replicon cell line resembling a persistent infection. On the other hand, translation from the encephalomyocarditis virus IRES is diminished in the presence of H2O2, suggesting that H2O2 translational responsiveness is a specific property of the HCV IRES and is not a general phenomenon for all viral IRESs. Altogether, these results suggest that HCV adapts to physio/pathological oxidative stress in the host cell by mediating La cytoplasmic shuttling to enhance its IRES-dependent translation.
Highlights
The hepatitis C virus (HCV) is a single-stranded, positivesense RNA virus belonging to the genus Hepacivirus of the family Flaviviridae (Chan, 2014)
Using Huh7 cells transiently transfected with the bicistronic reporter pRL1b mRNA in which translation of the Renilla luciferase and firefly luciferase is driven by cap-dependent and HCV internal ribosome entry site (IRES)-dependent translation, respectively, we have previously shown that H2O2 stimulates translation from the HCV IRES in tissue-cultured cells (MacCallum et al, 2006)
We showed that translation from the HCV IRES was upregulated by a range of physio/pathological levels of H2O2 that could be encountered by the virus during its prolonged phase of chronic infection, which has significance to HCV survival in an environment with continuously fluctuating levels of oxidants
Summary
The hepatitis C virus (HCV) is a single-stranded, positivesense RNA virus belonging to the genus Hepacivirus of the family Flaviviridae (Chan, 2014). Its 9.6 kb genome encodes a single polypeptide, which is cleaved by the host and viral proteases into structural proteins core, envelopes E1 and E2, and non-structural proteins p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. HCV poses a major threat to human health, causing chronic hepatitis, which can progress into cirrhosis and/or hepatocellular carcinoma. There is accumulating evidence that oxidative stress may be responsible for the pathogenesis of viral hepatitis and other forms of liver diseases (Paracha et al, 2013). Oxidative stress is a prominent clinical feature associated with HCV infection. Proteomic and microarray analysis of liver biopsies revealed increased oxidative stress in hepatitis C samples (Diamond et al, 2007; Yamashita et al, 2001)
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