title: High-throughput identification of loss-of-function mutations for anti-interferon activity in the influenza A virus NS segment.
abstract: Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated.
  
  
  OBJECTIVE
  Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (∼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA Molecular Biology Institute, University of California, Los Angeles, California, USA.
authors: Nicholas C
authors: NC
authors: Wu
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Arthur P
authors: AP
authors: Young
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Laith Q
authors: LQ
authors: Al-Mawsawi
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: C Anders
authors: CA
authors: Olson
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Jun
authors: J
authors: Feng
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Hangfei
authors: H
authors: Qi
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Harding H
authors: HH
authors: Luan
authors: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Xinmin
authors: X
authors: Li
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors: Ting-Ting
authors: TT
authors: Wu
authors: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA Molecular Biology Institute, University of California, Los Angeles, California, USA AIDS Institute, University of California, Los Angeles, California, USA RSun@mednet.ucla.edu.
authors: Ren
authors: R
authors: Sun
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D009980:Influenza A virus / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000502:physiology*; D007370:Interferon Type I / Q000037:antagonists & inhibitors*; D008967:Molecular Biology / Q000379:methods; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000172:deficiency* / Q000378:metabolism*; D014773:Virology / Q000379:methods
chemical_list: D007370:Interferon Type I; C108309:NS protein, influenza virus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins