Fig 8.

Specific interaction between miR-122 and the 5′ UTR of HCV is required for HCV replication. (A) Structures of pri-miR-122 and the nucleotide sequences of WT and MT miR-122, which has a substitution of uridine to adenosine in the seed domain and an additional complementary substitution of adenosine to uridine for stable expression. (B) WT or MT miR-122 was introduced into Hec1B cells by a lentiviral vector, and miR-122 expression levels were determined by qRT-PCR. (C) HCVcc was inoculated into Hec1B cells expressing either WT or MT miR-122 and control cells at an MOI of 1, and the intracellular HCV RNA levels were determined by qRT-PCR. (D) Diagram of mutant viruses HCVcc-M1 and HCVcc-M2 carrying complementary substitutions in the miR-122-binding site 1 alone and both sites 1 and 2 in the 5′ UTR of HCV, respectively. (E) Viral RNA of HCVcc, HCVcc-M1, or HCVcc-M2 was electroporated into Huh7.5.1 cells expressing either WT or MT miR-122, and infectious titers of the viruses recovered in the culture supernatants at 72 h postinfection of the second passage were determined by a focus-forming assay in cells expressing either WT or MT miR-122. Red, blue, and green bars, infectious titers of HCVcc, HCVcc-M1, and HCV-M2, respectively. (F) HCVcc, HCVcc-M1, or HCV-M2 was inoculated into Hec1B cells expressing either or both WT and MT miR-122 at an MOI of 0.5, and intracellular HCV RNA levels were determined at 12, 24, and 36 h postinfection by qRT-PCR. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control cells.