High-Throughput Identification of Loss-of-Function Mutations for Anti-Interferon Activity in the Influenza A Virus NS Segment

Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a sys-tematic approach to screen for loss-of-functionmutations 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 in-terferon, 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-functionmutations in the influenza A virus NS segment that were sensi-tive 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. IMPORTANCE 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 occur-rence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advan