MiR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5 UTR RNA to stabilize and promote viral RNA accumulation

Abstract

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5 UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5 untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5 UTR RNA folding, and mutant 5 UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5 UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5 end annealing or 3 overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5 UTR, in conjunction with any Ago isoform, modifies the 5 UTR structure to stabilize the viral genome and promote HCV RNA accumulation.