WU NC, Young AP, Al-Mawsawi LQ, Olson CA, et al. High-throughput identification of loss-of-function mutations for anti-interferon activity in influenza A virus NS segment. J Virol. 2014 Jun 25. pii: JVI.01494-14.
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 fitness disadvantage in a specified growth condition. Our methodology is 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 on 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.
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