Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2α under oxidative stress

Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H 2 O 2 ) as a stress inducer. In H 2 O 2 -sensitive HeLa cells, H 2 O 2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2 α phosphorylation. A phosphomimetic of eIF2 α , which mimics the structure of the phosphorylated eIF2 α , was sufficient to repress translation in the absence of H 2 O 2 . In H 2 O 2 -resistant HepG2 cells, H 2 O 2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2 α . It was postulated that H 2 O 2 might stimulate translation in HepG2 cells via an eIF2 α -independent mechanism, whereas the simultaneous phosphorylation of eIF2 α repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2 α -SA, resulting in further enhancement of both translational activities after exposure to H 2 O 2 . In HuH7 cells, which exhibited an intermediate level of sensitivity towards H 2 O 2 , both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H 2 O 2 , but the highest level of induction was achieved with a low level of H 2 O 2 , which may represent the physiological level of H 2 O 2 . At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.