Hepatitis C virus functionally sequesters miR-122 [RNA-Seq]

Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5’UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect could be relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and number of sites. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV. mRNA-seq libraries were generated from mock or J6/JFH1 Clone2 infected Huh7.5 cells. Cells were infected at an MOI of 1-2 and harvested at 72 hours and 96 hours post-infection for CLIP. Libraries were generated using Illumina Truseq technology.

丙型肝炎病毒（Hepatitis C virus, HCV）独特依赖肝脏特异性微RNA-122（microRNA-122, miR-122）完成复制，但目前尚未探索其感染过程中对内源性微RNA靶点的全局调控效应。本研究针对HCV感染过程中的人源Argonaute（Ago）开展高通量测序与交联免疫沉淀（high-throughput sequencing and crosslinking immunoprecipitation, HITS-CLIP）实验，结果显示HCV 5'非翻译区（5'UTR）在已知及预测的miR-122结合位点处存在强烈的Ago结合信号。在人类转录组层面，我们观察到病毒感染期间，miR-122靶点的Ago结合水平显著降低，且功能性mRNA发生去抑制现象。这种miR-122“海绵”效应可通过改变病毒的微RNA嗜性得到缓解，并重定向至miR-15靶点。携带miR-122结合位点的报告基因单细胞表达数据表明，HCV感染引发的去抑制效应显著，且其程度依赖于靶点的表达水平与结合位点数量。我们构建了HCV诱导的miR-122隔离定量数学模型，并提出HCV RNA对miR-122的此种抑制作用，可能会导致宿主所有miR-122靶点发生全局去抑制，从而为HCV的长期致癌潜能创造适宜的细胞环境。本研究从模拟感染（mock）或J6/JFH1 Clone2感染的Huh7.5细胞中构建mRNA测序文库。感染采用的感染复数（multiplicity of infection, MOI）为1~2，分别于感染后72小时与96小时收集样本用于CLIP实验。测序文库采用Illumina Truseq技术构建。