title: Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir.
abstract: MicroRNA 122 (miR-122) stimulates the replication and translation of hepatitis C virus (HCV) RNA by binding to two adjacent sites, S1 and S2, within the HCV 5'UTR. We demonstrated previously that the miR-122 antagomir miravirsen (SPC3649) suppresses the infection of HCV strain JFH1-based recombinants with HCV genotypes 1-6 5'UTR-NS2 in human hepatoma Huh7.5 cells. However, specific S1 mutations were permitted and conferred virus resistance to miravirsen treatment. Here, using the J6 (genotype 2a) 5'UTR-NS2 JFH1-based recombinant, we performed reverse-genetics analysis of S1 (ACACUCCG, corresponding to miR-122 seed nucleotide positions 8-1), S2 (CACUCC, positions 7-2), and ACCC (positions 1-4) at the 5' end of the HCV genome (5'E); the CC at positions 2-3 of 5'E is involved in miR-122 binding. We demonstrated that the 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 was permitted. In S1 and S2, several single mutations were allowed at specific positions. A UCC → CGA change at positions 4-3-2 of S1, S2, or both S1 and S2 (S1/S2), as well as a C → G change at position 2 of S1/S2 were permitted. We found that 5'E mutations did not confer virus resistance to miravirsen treatment. However, mutations in S1 and S2 induced virus 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 virus escape to host-targeting antiviral approaches.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Hvidovre, Denmark; Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.|Institute of Human Virology and Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Li
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Hvidovre, Denmark; Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Long
authors.initials: L
authors.lastname: Van Pham
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Hvidovre, Denmark; Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Uzcategui
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital, Hvidovre, Denmark; Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D020121:5' Untranslated Regions; D000070416:Antagomirs / Q000378:metabolism*; D024882:Drug Resistance, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D035683:MicroRNAs / Q000378:metabolism*; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D059386:Reverse Genetics; D014779:Virus Replication
chemical_list: D020121:5' Untranslated Regions; D000070416:Antagomirs; C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D012367:RNA, Viral