Hopcraft Sharon E, Azarm Kristopher D, Israelow Benjamin, Lévêque Nicolas, Schwarz Megan C, Hsu Tien-Huei, Chambers Matthew T, Sourisseau Marion, Semler Bert L, Evans Matthew J
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Clinical and Molecular Virology Unit (EA-4684 CardioVir), School of Medicine, University of Reims Champagne-Ardenne, Reims, France; Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, California, USA.
mSphere. 2015 Nov 25;1(1). doi: 10.1128/mSphere.00009-15. eCollection 2016 Jan-Feb.
Hepatitis C virus (HCV) replication requires binding of the liver-specific microRNA (miRNA) miR-122 to two sites in the HCV 5' untranslated region (UTR). Although we and others have shown that viral genetics impact the amount of active miR-122 required for replication, it is unclear if HCV can replicate in the complete absence of this miRNA. To probe the absolute requirements for miR-122 and the genetic basis for those requirements, we used clustered regularly interspaced short palindromic repeat (CRISPR) technology to knock out miR-122 in Huh-7.5 cells and reconstituted these knockout (KO) cells with either wild-type miR-122 or a mutated version of this miRNA. We then characterized the replication of the wild-type virus, as well as a mutated HCV bearing 5' UTR substitutions to restore binding to the mutated miR-122, in miR-122 KO Huh-7.5 cells expressing no, wild-type, or mutated miR-122. We found that while replication was most efficient when wild-type or mutated HCV was provided with the matched miR-122, inefficient replication could be observed in cells expressing the mismatched miR-122 or no miR-122. We then selected viruses capable of replicating in cells expressing noncognate miR-122 RNAs. Unexpectedly, these viruses contained multiple mutations throughout their first 42 nucleotides that would not be predicted to enhance binding of the provided miR-122. These mutations increased HCV RNA replication in cells expressing either the mismatched miR-122 or no miR-122. These data provide new evidence that HCV replication can occur independently of miR-122 and provide unexpected insights into how HCV genetics influence miR-122 requirements. IMPORTANCE Hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. HCV infection requires miR-122, which is expressed only in liver cells, and thus is one reason that replication of this virus occurs efficiently only in cells of hepatic origin. To understand how HCV genetics impact miR-122 usage, we knocked out miR-122 using clustered regularly interspaced short palindromic repeat (CRISPR) technology and adapted virus to replicate in the presence of noncognate miR-122 RNAs. In doing so, we identified viral mutations that allow replication in the complete absence of miR-122. This work provides new insights into how HCV genetics influence miR-122 requirements and proves that replication can occur without this miRNA, which has broad implications for how HCV tropism is maintained.
丙型肝炎病毒（HCV）的复制需要肝脏特异性微小RNA（miRNA）miR - 122与HCV 5'非翻译区（UTR）中的两个位点结合。尽管我们和其他人已经表明病毒遗传学影响复制所需的活性miR - 122的量，但尚不清楚HCV在完全没有这种miRNA的情况下是否能够复制。为了探究miR - 122的绝对需求以及这些需求的遗传基础，我们使用成簇规律间隔短回文重复序列（CRISPR）技术在Huh - 7.5细胞中敲除miR - 122，并用野生型miR - 122或该miRNA的突变版本重建这些敲除（KO）细胞。然后，我们在不表达、表达野生型或突变型miR - 122的miR - 122 KO Huh - 7.5细胞中，对野生型病毒以及携带5' UTR替代突变以恢复与突变型miR - 122结合的突变型HCV的复制进行了表征。我们发现，当野生型或突变型HCV与匹配的miR - 122一起提供时，复制效率最高，而在表达不匹配的miR - 122或不表达miR - 122的细胞中可观察到低效复制。然后，我们选择了能够在表达非同源miR - 122 RNA的细胞中复制的病毒。出乎意料的是，这些病毒在其最初的42个核苷酸中含有多个突变，预计这些突变不会增强所提供的miR - 122的结合。这些突变增加了在表达不匹配的miR - 122或不表达miR - 122的细胞中的HCV RNA复制。这些数据提供了新的证据，表明HCV复制可以独立于miR - 122发生，并为HCV遗传学如何影响miR - 122需求提供了意想不到的见解。重要性丙型肝炎病毒（HCV）是西半球肝癌的主要病因。HCV感染需要miR - 122，而miR - 122仅在肝细胞中表达，因此这是该病毒仅在肝源性细胞中高效复制的原因之一。为了了解HCV遗传学如何影响miR - 122的使用，我们使用成簇规律间隔短回文重复序列（CRISPR）技术敲除miR - 122，并使病毒适应在非同源miR - 122 RNA存在的情况下复制。通过这样做，我们鉴定出了允许在完全没有miR - 122的情况下复制的病毒突变。这项工作为HCV遗传学如何影响miR - 122需求提供了新的见解，并证明了没有这种miRNA也可以发生复制，这对维持HCV嗜性具有广泛的意义。
