Grant: Discovery Grants Program - Individual

Hepatitis C virus (HCV), a positive-sense, single-stranded RNA virus, is a member of the Hepacivirus genus in the Flaviviridae family. Hepaciviruses with close homology to HCV have been isolated in recent years from numerous animal species, such as dog, horse, bat, and cattle. As such, basic research on HCV is not only critical for understanding HCV biology, it also serves as an excellent model virus for understanding the biology of animal hepaciviruses. The long-term goal of this research program is to study the molecular mechanisms of viral translation modulation using HCV as a model. During the current NSERC Discovery grant funding period (2013-2018), we are investigating the regulatory role of non-structural protein-5A (NS5A) in HCV RNA translation and the function of the viral 3untranslated region (3UTR). We showed that NS5A downregulates HCV translation. The poly(U/UC) sequence in the viral 3UTR is required for translation downregulation by NS5A. Detailed mapping experiments demonstrated that amino acid R112 in domain I of NS5A, K312 in domain II, and E446 in domain III are involved in translation downregulation. Mechanistically, we showed that mutating R112 in NS5A domain I abrogates its binding to poly(U/UC) RNA, suggesting that NS5A - poly(U/UC) RNA interaction may represent a mechanism of how NS5A modulates viral translation. We also studied the effects of the PI3K-Akt pathway on HCV RNA translation. We found that this pathway increases HCV translation with the involvement of all three Akt isoforms. The research, performed by three graduate students and one post-doctoral fellow, resulted in five publications. Based on our ongoing research, the short-term goal of this proposal is to further characterize the molecular mechanisms by which HCV NS5A modulates viral RNA translation. We will test the hypothesis that HCV NS5A protein regulates HCV RNA translation through multiple mechanisms in five objectives. 1. Characterize the role of the variable and X regions in the HCV 3UTR in viral RNA translation modulation by NS5A. 2. Characterize the effect of NS5A dimerization on HCV RNA translation. 3. Characterize the effect of NS5A hyper-phosphorylation on HCV RNA translation. 4. Characterize the molecular mechanisms by which HCV proteins and cellular proteins modulate the effect of NS5A on HCV RNA translation. 5. To study the role of the MAPK signal transduction pathway in HCV translation modulation by NS5A. This proposal will continue our research program on viral translation modulation using HCV as a model. Our research will further determine how an HCV protein regulates viral translation. As such, knowledge gained from this proposal will also help understand protein translation modulation of related animal hepaciviruses. The proposed research will be carried out by graduate students.