Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir
Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir
复制标题
丙型肝炎病毒 microRNA-122 结合序列的功能分析以及对特定 antagomir 高耐药性变体的鉴定
DOI：
10.1099/jgv.0.000445
复制
发表时间：
2016-06-01
影响因子：
3.8
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
--
作者：
Li, Yi-Ping;Long Van Pham;Bukh, Jens
MicroRNA 122 （miR-122）通过结合HCV 5'UTR内的两个相邻位点S1和S2来刺激丙型肝炎病毒（HCV） RNA的复制和翻译。我们之前证明，miR-122 antagomir miravirsen （SPC3649）抑制HCV基因型为1-6 5'UTR-NS2的HCV菌株jfh1重组体在人肝癌Huh7.5细胞中的感染。然而，特定的S1突变是允许的，并赋予病毒对miravirsen治疗的抗性。在这里，我们使用基于J6（基因型2a） 5‘ utr - ns2 jfh1的重组基因，对HCV基因组（5e） 5’端的S1 （ACACUCCG，对应miR-122种子核苷酸位置8-1）、S2 （CACUCC，位置7-2）和ACCC（位置1-4）进行了反向遗传学分析；5e位点2-3处的CC参与了miR-122的结合。我们证明了5'E需要4个核苷酸才能达到最佳功能，并且在5'E的3号位置上允许G或A或在2-3号位置上允许组合GA。在S1和S2中，在特定位置允许几个单突变。允许在S1、S2或S1和S2 （S1/ S2）的4-3-2位置发生UCC -> CGA变化，以及在S1/ S2的2位置发生C - >g变化。我们发现5e突变没有赋予病毒对miravirsen治疗的抗性。然而，S1和S2的突变诱导了病毒抗性，并且S1和/或S2的联合突变比单一突变具有更高的抗性。鉴定miR-122抗塔戈莫耐药相关突变将有助于研究miR-122在HCV生命周期中的其他功能以及病毒逃逸到靶向宿主抗病毒方法的机制。
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.