HCV IRES-dependent translation under stress conditions

Abstract

The regulation of translation is an important control mechanism in gene expression but is still not completely understood. However, it is becoming increasingly evident that the regulation of translation provides the cell with the plasticity that is needed to respond to rapid changes in the environment. Such regulation is very important under certain conditions - such as cellular stress (for example, heat shock, hypoxia, nutrient deprivation and ER stress) - that require immediate changes in protein levels. Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the a-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNAi) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2a phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNAi ternary complex. Here, I report that eIF2A, an alternative initiator tRNA-binding protein, plays a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2a phosphorylation by activating the eIF2a kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV IRES is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNAi to the P site of the 40S ribosomal subunit. In addition, binding of Met-tRNAi to 40S ribosome was strongly enhanced by addition of eIF5B. eIF5B also promotes binding of the initiator tRNA to ribosomes and it couples the ribosomal subunits, in spite of the fact that, in contrast to IF2, no direct binding of eIF5B to initiator tRNA has been reported. I found that eIF2A binds to eIF5B directly. Notably, my data provide biochemical evidence that eIF2A recruits initiator tRNA to the 40S subunit and eIF5B stabilizes tRNA binding to the 40S subunit. Therefore, eIF2A and eIF5B cooperatively facilitates binding of Met-tRNAi to 40S ribosome in a HCV IRES-dependent translation. The study of HCV IRES-dependent translation by eIF2A and eIF5B will add a new dimension to our understanding of translational regulation under different physiological conditions.