TY - JOUR T1 - Accurate measurement of the effects of all amino-acid mutations to influenza hemagglutinin JF - bioRxiv DO - 10.1101/047571 SP - 047571 AU - Michael B. Doud AU - Jesse D. Bloom Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/04/07/047571.abstract N2 - 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. ER -