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

TY - JOUR

T1 - Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against specific antagomir

AU - Li, Yi-ping

AU - Pham, Long

AU - Uzcategui, Nathalie

AU - Bukh, Jens

PY - 2016/6

Y1 - 2016/6

N2 - 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 5ʹUTR. We demonstrated previously that the miR-122 antagomir miravirsen (SPC3649) suppresses the infection of HCV strain JFH1-based recombinants with HCV genotypes 1–6 5ʹUTR–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) 5ʹUTR–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 5ʹ end of the HCV genome (5ʹE); the CC at positions 2–3 of 5ʹE is involved in miR-122 binding. We demonstrated that the 5ʹE required four nucleotides for optimal function, and that G or A at position 3 or combined GA at positions 2–3 of 5ʹE 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 5ʹE 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.

AB - 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 5ʹUTR. We demonstrated previously that the miR-122 antagomir miravirsen (SPC3649) suppresses the infection of HCV strain JFH1-based recombinants with HCV genotypes 1–6 5ʹUTR–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) 5ʹUTR–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 5ʹ end of the HCV genome (5ʹE); the CC at positions 2–3 of 5ʹE is involved in miR-122 binding. We demonstrated that the 5ʹE required four nucleotides for optimal function, and that G or A at position 3 or combined GA at positions 2–3 of 5ʹE 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 5ʹE 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.

U2 - 10.1099/jgv.0.000445

DO - 10.1099/jgv.0.000445

M3 - Journal article

C2 - 26935756

SN - 0022-1317

VL - 97

SP - 1381

EP - 1394

JO - Journal of General Virology

JF - Journal of General Virology

ER -