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.