PT  - JOURNAL ARTICLE
AU  - Michael B. Doud
AU  - Jesse D. Bloom
TI  - Accurate measurement of the effects of all amino-acid mutations to influenza hemagglutinin
AID  - 10.1101/047571
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 047571
4099  - http://biorxiv.org/content/early/2016/04/07/047571.short
4100  - http://biorxiv.org/content/early/2016/04/07/047571.full
AB  - Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We previously used high-throughput mutagenesis and deep sequencing to estimate the effects of all mutations to an H1 influenza hemagglutinin on viral replication in cell culture (Thyagarajan and Bloom, 2014); however, these measurements suffered from sub-stantial noise. Here we describe advances that greatly improve the accuracy and reproducibility of our measurements. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. However, other regions – including stalk epitopes targeted by broadly neutralizing antibodies – have a limited capacity to evolve. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.