Michael A. Palmer, University of Saskatchewan

Michael A. Palmer, Chelsey D. Thistle, Yalena Amador-Cañizares, Joyce A. Wilson (Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, SK, Canada)

Expressing & Multiplying

Hepatitis C virus (HCV), a positive-sense, single-stranded RNA virus, relies on microRNA miR-122 annealing within its 5'UTR to promote viral replication. Typically, miRNAs bind the 3'UTR of mRNA, suppressing translation and increasing RNA decay through a process known as RNA interference (RNAi). By contrast, miR-122 binds two sites within the HCV 5'UTR, promoting genome stability, genomic RNA accumulation, and viral translation. During RNAi, Argonaute 2 (Ago2) uses miRNAs to target mRNA, relying on functional domains and post-translational modifications to regulate processes including miRNA loading, miRNA strand selection, and protein complex assembly. While Ago2 is also required for miR-122 promotion of HCV, its specific roles are unknown. Using an Ago2 trans-complementation system, we showed Ago2's interaction with TNRC6, a protein essential for RNAi, is also required for HCV replication and translation promotion; conversely, Ago2 mutants unable to cleave, unwind RNA, or interact with other RNAi components still supported viral replication, suggesting these activities are not essential for miRNA promotion of HCV. Further, several phosphorylation mutants affected Ago2's ability to promote HCV. Phosphorylation of serine-798 supported viral replication but not translation, while its dephosphorylation promoted viral translation over replication. This suggests a possible role for Ago2 phosphorylation in regulating the switch between HCV genome replication and translation. Previous studies indicate Ago2 phosphorylation alters its localization to cellular P-bodies, a potential mechanism underlying our observations which we are actively exploring. Altogether, we demonstrate Ago2's role in miR-122-dependent HCV promotion is complex and may modulate the balance between virus replication and translation.