Ono Chikako, Fukuhara Takasuke, Motooka Daisuke, Nakamura Shota, Okuzaki Daisuke, Yamamoto Satomi, Tamura Tomokazu, Mori Hiroyuki, Sato Asuka, Uemura Kentaro, Fauzyah Yuzy, Kurihara Takeshi, Suda Takahiro, Nishio Akira, Hmwe Su Su, Okamoto Toru, Tatsumi Tomohide, Takehara Tetsuo, Chayama Kazuaki, Wakita Takaji, Koike Kazuhiko, Matsuura Yoshiharu
Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
PLoS Pathog. 2017 May 11;13(5):e1006374. doi: 10.1371/journal.ppat.1006374. eCollection 2017 May.
miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5'UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.
miR-122是一种肝脏特异性微小RNA，是丙型肝炎病毒（HCV）感染肝脏嗜性的决定因素之一。虽然miR-122是HCV有效增殖所必需的，但我们之前已经表明，HCV在miR-122缺陷细胞中以低速率复制，这表明HCV-RNA能够以不依赖miR-122的方式进行增殖。我们在此使用miR-122敲除的Huh7（Huh7-122KO）细胞研究了miR-122在HCV-RNA复制和感染性颗粒产生中的作用。在HCV感染后，在Huh7-122KO细胞中观察到细胞内HCV-RNA水平和培养上清液中感染滴度略有增加。此外，在miR-122敲除的Huh7.5.1细胞中对HCV进行连续传代后，我们获得了一种适应性突变体HCV122KO，其在HCV基因2a型JFH1基因组的5'UTR中具有G28A替换，并且这种突变体可能有助于增强复制复合物的形成，多核糖体分析支持了这一可能性。我们还发现在基因型1b/2a嵌合病毒中，原本在核苷酸位置29处为腺嘌呤的miR-122结合位点周围引入了适应性突变。HCV122KO在miR-122敲除细胞和非肝细胞中无需外源性表达miR-122即可高效进行RNA复制。竞争试验表明，G28A突变体在不存在miR-122时占主导地位，但其在存在miR-122时的作用与野生型相当，这表明G28A突变在富含miR-122的肝细胞中不具有增殖优势。这些观察结果可能解释了临床发现，即miR-122缺陷的外周血单核细胞（PBMC）中G28A突变的阳性率高于患者血清，患者血清中主要包含来自HCV基因2a型患者的肝细胞衍生病毒。这些结果表明，能够以不依赖miR-122的方式在非肝细胞中增殖的HCV突变体的出现可能参与了慢性丙型肝炎患者肝外表现的诱导。
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