A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins

ABSTRACT Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes t...

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Autores principales:	Roland Remenyi, Hangfei Qi, Sheng-Yao Su, Zugen Chen, Nicholas C. Wu, Vaithilingaraja Arumugaswami, Shawna Truong, Virginia Chu, Tamar Stokelman, Hung-Hao Lo, C. Anders Olson, Ting-Ting Wu, Shu-Hwa Chen, Chung-Yen Lin, Ren Sun
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/124d6b95833940a0bd99d5b59e2a7974
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spelling	oai:doaj.org-article:124d6b95833940a0bd99d5b59e2a79742021-11-15T15:45:54ZA Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins10.1128/mBio.01469-142150-7511https://doaj.org/article/124d6b95833940a0bd99d5b59e2a79742014-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01469-14https://doaj.org/toc/2150-7511ABSTRACT Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains. IMPORTANCE Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.Roland RemenyiHangfei QiSheng-Yao SuZugen ChenNicholas C. WuVaithilingaraja ArumugaswamiShawna TruongVirginia ChuTamar StokelmanHung-Hao LoC. Anders OlsonTing-Ting WuShu-Hwa ChenChung-Yen LinRen SunAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 5 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Roland Remenyi Hangfei Qi Sheng-Yao Su Zugen Chen Nicholas C. Wu Vaithilingaraja Arumugaswami Shawna Truong Virginia Chu Tamar Stokelman Hung-Hao Lo C. Anders Olson Ting-Ting Wu Shu-Hwa Chen Chung-Yen Lin Ren Sun A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins
description	ABSTRACT Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains. IMPORTANCE Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.
format	article
author	Roland Remenyi Hangfei Qi Sheng-Yao Su Zugen Chen Nicholas C. Wu Vaithilingaraja Arumugaswami Shawna Truong Virginia Chu Tamar Stokelman Hung-Hao Lo C. Anders Olson Ting-Ting Wu Shu-Hwa Chen Chung-Yen Lin Ren Sun
author_facet	Roland Remenyi Hangfei Qi Sheng-Yao Su Zugen Chen Nicholas C. Wu Vaithilingaraja Arumugaswami Shawna Truong Virginia Chu Tamar Stokelman Hung-Hao Lo C. Anders Olson Ting-Ting Wu Shu-Hwa Chen Chung-Yen Lin Ren Sun
author_sort	Roland Remenyi
title	A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins
title_short	A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins
title_full	A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins
title_fullStr	A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins
title_full_unstemmed	A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins
title_sort	comprehensive functional map of the hepatitis c virus genome provides a resource for probing viral proteins
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/124d6b95833940a0bd99d5b59e2a7974
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A Comprehensive Functional Map of the Hepatitis C Virus Genome Provides a Resource for Probing Viral Proteins

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