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Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against specific antagomir

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MicroRNA miR-122 stimulates the replication and translation of hepatitis C virus (HCV) RNA through binding to two adjacent sites S1 and S2 within the HCV 5' untranslated region (5'UTR). We previously demonstrated that the miR-122 antagomir miravirsen (SPC3649) suppressed the infection of JFH1-based recombinants with HCV genotype 1-6 5'UTR-NS2 in human hepatoma Huh7.5 cells. However, specific S1 mutations were permitted and conferred viral resistance to miravirsen treatment. Using the J6 (genotype 2a) 5'UTR-NS2 JFH1-based recombinant, we here performed reverse-genetics analysis of S1 (ACACUCCG, corresponding to miR-122 seed nucleotide positions 8-1), S2 (CACUCC, positions 7-2), and ACCC (position 1-4) at the 5'-end of its genome (5'E); the "CC" at positions 2-3 of 5'E is involved in miR-122 binding. We demonstrated that 5'E required four nucleotides for optimal function, and that G or A at position 3 or combined GA at positions 2-3 of 5'E were permitted. In S1 and S2, several single mutations were allowed at specific positions. UCC to CGA change at position 4-3-2 of S1, S2, or both S1 and S2 (S1/S2), as well as C to G change at position 2 of S1/S2 were permitted. We found that 5'E mutations did not confer viral resistance to miravirsen treatment. However, mutations in S1 and S2 induced viral resistance, and combined S1 and/or S2 mutations conferred higher resistance than single mutations. Identification of miR-122 antagomir resistance-associated mutations will facilitate the study of additional functions of miR-122 in the HCV life-cycle and the mechanism of viral escape to host-targeting antiviral approaches.
OriginalsprogEngelsk
TidsskriftJournal of General Virology
Vol/bind97
Sider (fra-til)1381-1394
ISSN0022-1317
DOI
StatusUdgivet - jun. 2016

ID: 157289778