Abstract
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.
Highlights
Seasonal influenza is a recurrent threat to human health, largely because it rapidly accumulates amino-acid mutations in proteins targeted by the immune system [1]
In our previous deep mutational scanning of influenza [4,5,6], we reduced this error rate by using overlapping paired-end reads
We have described new techniques that greatly improve the reproducibility of deep mutational scanning of influenza
Summary
Seasonal influenza is a recurrent threat to human health, largely because it rapidly accumulates amino-acid mutations in proteins targeted by the immune system [1]. We and others have used deep mutational scanning to estimate the effects of all amino-acid [4,5,6] or nucleotide [7,8] mutations to several influenza genes, and Heaton and coworkers [9] have used a similar approach to examine influenza’s tolerance to short insertions. These studies suffered from substantial noise that degrades the utility of their results. In every study that reported the results for independent experimental replicates, the replicate-to-replicate correlation was mediocre
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