Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants

Abstract

Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HA's functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino-acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA, and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature -but only when the measurements are made on a viral strain similar to the ones being studied in nature.influenza virus | hemagglutinin | deep mutational scanning | antigenic drift | epistasis S easonal H3N2 influenza virus evolves rapidly, fixing 3 to 4 amino-acid mutations per year in its hemagglutinin (HA) surface protein (1, 2). Many of these mutations contribute to the rapid antigenic drift that necessitates frequent updates to the annual influenza vaccine (3). This evolution is further characterized by competition and turnover among groups of strains called clades bearing different complements of mutations (4-8). Clades vary widely in their evolutionary success, with some dying out soon after emergence and others going on to take over the virus population. Several lines of evidence indicate that successful clades have higher fitness than clades that remain at low frequency (4-6, 9). A key goal in the study of H3N2 evolution is to identify the features that enable certain clades to succeed as others die out.Two main characteristics distinguish evolutionarily successful clades from their competitors: greater antigenic change, and efficient viral growth and transmission. In principle, experiments could be informative for identifying how mutations affect these features. Most work on influenza evolution to date has utilized experimental data to assess the antigenicity of circulating strains (11)(12)(13)(14)(15)(16). However, the non-antigenic effects of mutations also play an important role (5,7,9,17). Specifically, due to influenza virus's high mutation rate (18)(19)(20) and lack of intra-segment recombination (21), deleterious mutations become linked to beneficial ones. The resulting accumulation of deleterious mutations can affect non-antigenic properties central...

Coupling adaptive molecular evolution to phylodynamics using fitness-dependent birth-death models

“…We used the Lumière implementation of the MFBD model to estimate the fitness effects of amino acid mutations in the hemagglutinin (HA) protein of human influenza virus subtype H3N2. In order to ensure our fitness estimates were directly comparable to the mutational fitness effects previously estimated by Lee et al [2018], we focused our analysis on viral samples in the same antigenic cluster as the A/Perth/16/2009 strain studied by Lee et al for two reasons. First, Lee et al [2018] showed that the fitness effects of amino acid mutations in HA vary depending on the genetic background, with greater fitness differences between more divergent strains.…”

“…In order to ensure our fitness estimates were directly comparable to the mutational fitness effects previously estimated by Lee et al [2018], we focused our analysis on viral samples in the same antigenic cluster as the A/Perth/16/2009 strain studied by Lee et al for two reasons. First, Lee et al [2018] showed that the fitness effects of amino acid mutations in HA vary depending on the genetic background, with greater fitness differences between more divergent strains. We therefore only considered strains with low genetic divergence from A/Perth/16/2009.…”

“…where π k,i is the relative preference for amino acid i at site k. To compute these fitness effects, we used the averaged relative amino acid preferences reported in Dataset S3 of Lee et al [2018].…”

“…We also applied the MFBD model to estimate the fitness effects of mutations in the hemagglutinin (HA) protein of human influenza virus subtype H3N2. Lee et al [2018] recently estimated the relative preference for each amino acid residue at all sites in the HA protein in cell culture using a reverse genetics approach known as deep mutational scanning (DMS). The fitness effect of mutating one amino acid to another is expected to correlate strongly with the relative preference for each amino acid in these experiments.…”

Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

“…A recent approach combines mutagenesis by PCR with degenerate (NNK) primers with in-cell recombination to create variation at specific residues in the tail fiber of T3 phage (Yehl et al 2019). By contrast, human viruses often have reverse genetics systems in place where comprehensive mutant libraries can be prepared for single genes or regions within a gene (Doud et al 2018, Wu andQi 2019) using replicative plasmids that encode whole viruses or viral components. Similarly, deep mutational scanning can be performed on plasmid-encoded phage proteins like the MS2 capsid protein (Hartman et al 2018).…”