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Open AccessArticle

Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin

by Michael B. Doud 1,2,3 and Jesse D. Bloom 1,2,*
Division of Basic Sciences Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA 98109, USA
Department of Genome Sciences, University of Washington, 3720 15th Ave NE, Seattle, WA 98195-5065, USA
Medical Scientist Training Program, University of Washington, Seattle, WA 98109, USA
Author to whom correspondence should be addressed.
Academic Editors: Eric O. Freed and Thomas Klimkait
Viruses 2016, 8(6), 155;
Received: 23 April 2016 / Revised: 21 May 2016 / Accepted: 25 May 2016 / Published: 3 June 2016
(This article belongs to the Special Issue Viruses 2016 - At the Forefront of Virus-Host Interactions)
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 and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin—including the stalk epitopes targeted by broadly neutralizing antibodies—have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution. View Full-Text
Keywords: influenza; hemagglutinin; mutational tolerance; deep mutational scanning; evolution influenza; hemagglutinin; mutational tolerance; deep mutational scanning; evolution
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MDPI and ACS Style

Doud, M.B.; Bloom, J.D. Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin. Viruses 2016, 8, 155.

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