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.

Location specific small RNA annealing to the HCV 5’ UTR promotes Hepatitis C Virus replication by favoring IRES formation and stimulating virus translation

“…However, simultaneous knock-down of these three enzymes cannot completely rescue HCV replication in the absence of miR-122, suggesting that other roles exist (23). miR-122 also promotes HCV translation and recent reports hypothesize that miR-122 modifies the HCV genome to induce the canonical 5’UTR IRES structure (Figure 1B and C) (12, 13, 24). Finally, miR-122 has been reported to directly induce genome amplification (25, 26).…”

“…miR-122 knockout (miR-122 KO) Huh-7.5 (33), Ago2 knockout (Ago2 KO) Huh-7.5 cells (13) and DROSHA/Ago2 KO cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 0.1 nM non-essential amino acids (Wisent, Montreal, Canada) and 100 ug/ml Pen/Strep (Invitrogen, Burlingtion, Canada). miR-122 knockout Huh-7.5 cells were a kind gift from Dr Matthew Evans.…”

“…SLII is divided into two parts, SLIIa which induces SLII to form a bent structure that directs SLIIb, to the ribosomal E-site in the head region of the 40S subunit, facilitating 80S ribosome assembly (7–9). The first 42 nucleotides on the 5’UTR are not considered part of the IRES and forms SLI and an RNA structured element created by annealing of two copies of microRNA-122 (miR-122), a host microRNA found in human liver cells (10–13). This tri-molecular structure is required for virus replication (11–14).…”

“…We previously showed that annealing of small interfering RNAs (siRNA) to the HCV 5’ UTR can mimic the pro-viral activity of miR-122 (13). Like miRNAs, siRNAs are also 21–23 nucleotides in length and associate with Ago proteins, but based on associating with Ago2 and perfect sequence match with their targets, induce mRNA cleavage and gene knockdown (28, 29).…”

“…That siRNA annealing promoted HCV, suggested that the complex annealing pattern formed by miR-122 on the HCV genome was not required for the pro-viral activity. It also provided a method to assess the impact of small RNA annealing to other locations on the genome on HCV replication (13). Using a panel of HCV genome-targeting siRNAs, we found that annealing between nucleotides 1 and 44 in HCV 5’UTR, promoted HCV replication, and annealing within the IRES, NS5B and 3’UTR regions did not.…”