Viral Determinants of miR-122-Independent Hepatitis C Virus Replication

ABSTRACT 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...

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Autores principales:	Sharon E. Hopcraft, Kristopher D. Azarm, Benjamin Israelow, Nicolas Lévêque, Megan C. Schwarz, Tien-Huei Hsu, Matthew T. Chambers, Marion Sourisseau, Bert L. Semler, Matthew J. Evans
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	hepatitis C virus microRNA miR-122 CRISPR Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ef738360aa8246348fe99a4903d25002
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spelling	oai:doaj.org-article:ef738360aa8246348fe99a4903d250022021-11-15T15:21:37ZViral Determinants of miR-122-Independent Hepatitis C Virus Replication10.1128/mSphere.00009-152379-5042https://doaj.org/article/ef738360aa8246348fe99a4903d250022016-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00009-15https://doaj.org/toc/2379-5042ABSTRACT 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.Sharon E. HopcraftKristopher D. AzarmBenjamin IsraelowNicolas LévêqueMegan C. SchwarzTien-Huei HsuMatthew T. ChambersMarion SourisseauBert L. SemlerMatthew J. EvansAmerican Society for Microbiologyarticlehepatitis C virusmicroRNAmiR-122CRISPRMicrobiologyQR1-502ENmSphere, Vol 1, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	hepatitis C virus microRNA miR-122 CRISPR Microbiology QR1-502
spellingShingle	hepatitis C virus microRNA miR-122 CRISPR Microbiology QR1-502 Sharon E. Hopcraft Kristopher D. Azarm Benjamin Israelow Nicolas Lévêque Megan C. Schwarz Tien-Huei Hsu Matthew T. Chambers Marion Sourisseau Bert L. Semler Matthew J. Evans Viral Determinants of miR-122-Independent Hepatitis C Virus Replication
description	ABSTRACT 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.
format	article
author	Sharon E. Hopcraft Kristopher D. Azarm Benjamin Israelow Nicolas Lévêque Megan C. Schwarz Tien-Huei Hsu Matthew T. Chambers Marion Sourisseau Bert L. Semler Matthew J. Evans
author_facet	Sharon E. Hopcraft Kristopher D. Azarm Benjamin Israelow Nicolas Lévêque Megan C. Schwarz Tien-Huei Hsu Matthew T. Chambers Marion Sourisseau Bert L. Semler Matthew J. Evans
author_sort	Sharon E. Hopcraft
title	Viral Determinants of miR-122-Independent Hepatitis C Virus Replication
title_short	Viral Determinants of miR-122-Independent Hepatitis C Virus Replication
title_full	Viral Determinants of miR-122-Independent Hepatitis C Virus Replication
title_fullStr	Viral Determinants of miR-122-Independent Hepatitis C Virus Replication
title_full_unstemmed	Viral Determinants of miR-122-Independent Hepatitis C Virus Replication
title_sort	viral determinants of mir-122-independent hepatitis c virus replication
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/ef738360aa8246348fe99a4903d25002
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Viral Determinants of miR-122-Independent Hepatitis C Virus Replication

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