title: 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 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Roland
authors.initials: R
authors.lastname: Remenyi
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Hangfei
authors.initials: H
authors.lastname: Qi
authors.forename: Sheng-Yao
authors.initials: SY
authors.lastname: Su
authors.affiliation: Department of Human Genetics, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Zugen
authors.initials: Z
authors.lastname: Chen
authors.affiliation: The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Wu
authors.forename: Vaithilingaraja
authors.initials: V
authors.lastname: Arumugaswami
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Shawna
authors.initials: S
authors.lastname: Truong
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Virginia
authors.initials: V
authors.lastname: Chu
authors.affiliation: Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Tamar
authors.initials: T
authors.lastname: Stokelman
authors.affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Hung-Hao
authors.initials: HH
authors.lastname: Lo
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: C Anders
authors.initials: CA
authors.lastname: Olson
authors.affiliation: Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Wu
authors.affiliation: Institute of Information Science, Academia Sinica, Taipei, Taiwan.
authors.forename: Shu-Hwa
authors.initials: SH
authors.lastname: Chen
authors.affiliation: Institute of Information Science, Academia Sinica, Taipei, Taiwan.
authors.forename: Chung-Yen
authors.initials: CY
authors.lastname: Lin
authors.affiliation: rsun@mednet.ucla.edu.
authors.forename: Ren
authors.initials: R
authors.lastname: Sun
mesh_terms: D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015723:Gene Library; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016254:Mutagenesis, Insertional; D010957:Plasmids; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004251:DNA Transposable Elements; D004279:DNA, Viral; D014764:Viral Proteins