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

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 mut...

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Autores principales: Doud, Michael B., Bloom, Jesse D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926175/
https://www.ncbi.nlm.nih.gov/pubmed/27271655
http://dx.doi.org/10.3390/v8060155
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author Doud, Michael B.
Bloom, Jesse D.
author_facet Doud, Michael B.
Bloom, Jesse D.
author_sort Doud, Michael B.
collection PubMed
description 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.
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spelling pubmed-49261752016-07-06 Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin Doud, Michael B. Bloom, Jesse D. Viruses Article 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. MDPI 2016-06-03 /pmc/articles/PMC4926175/ /pubmed/27271655 http://dx.doi.org/10.3390/v8060155 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Doud, Michael B.
Bloom, Jesse D.
Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin
title Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin
title_full Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin
title_fullStr Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin
title_full_unstemmed Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin
title_short Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin
title_sort accurate measurement of the effects of all amino-acid mutations on influenza hemagglutinin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926175/
https://www.ncbi.nlm.nih.gov/pubmed/27271655
http://dx.doi.org/10.3390/v8060155
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