Abstract:Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indi… Show more
“…Unfortunately, it is not straightforward to distinguish between these two explanations. This difficulty in pinpointing reasons for inter-study variation highlights a limitation of the high-throughput experimental methodology employed by ourselves and Wu et al (2014): while such experiments provide a wealth of data, numerous factors can create noise in these data (sequencing errors, population bottlenecks, epistasis among mutations, etc ). Realizing the full potential of such studies will therefore require extensive experimental controls and biological replicates to quantify errors and noise to enable comparisons across data sets.10.7554/eLife.03300.015Figure 7.Correlation of the site-specific amino-acid preferences determined in our study with the “relative fitness” (RF) values reported by Wu et al (2014).…”
Influenza is notable for its evolutionary capacity to escape immunity targeting the viral hemagglutinin. We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability. We created mutant viruses that incorporate most of the ≈104 amino-acid mutations to hemagglutinin from A/WSN/1933 (H1N1) influenza. After passaging these viruses in tissue culture to select for functional variants, we used deep sequencing to quantify mutation frequencies before and after selection. These data enable us to infer the preference for each amino acid at each site in hemagglutinin. These inferences are consistent with existing knowledge about the protein's structure and function, and can be used to create a model that describes hemagglutinin's evolution far better than existing phylogenetic models. We show that hemagglutinin has a high inherent tolerance for mutations at antigenic sites, suggesting that this is one factor contributing to influenza's antigenic evolution.DOI:
http://dx.doi.org/10.7554/eLife.03300.001
“…Unfortunately, it is not straightforward to distinguish between these two explanations. This difficulty in pinpointing reasons for inter-study variation highlights a limitation of the high-throughput experimental methodology employed by ourselves and Wu et al (2014): while such experiments provide a wealth of data, numerous factors can create noise in these data (sequencing errors, population bottlenecks, epistasis among mutations, etc ). Realizing the full potential of such studies will therefore require extensive experimental controls and biological replicates to quantify errors and noise to enable comparisons across data sets.10.7554/eLife.03300.015Figure 7.Correlation of the site-specific amino-acid preferences determined in our study with the “relative fitness” (RF) values reported by Wu et al (2014).…”
Influenza is notable for its evolutionary capacity to escape immunity targeting the viral hemagglutinin. We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability. We created mutant viruses that incorporate most of the ≈104 amino-acid mutations to hemagglutinin from A/WSN/1933 (H1N1) influenza. After passaging these viruses in tissue culture to select for functional variants, we used deep sequencing to quantify mutation frequencies before and after selection. These data enable us to infer the preference for each amino acid at each site in hemagglutinin. These inferences are consistent with existing knowledge about the protein's structure and function, and can be used to create a model that describes hemagglutinin's evolution far better than existing phylogenetic models. We show that hemagglutinin has a high inherent tolerance for mutations at antigenic sites, suggesting that this is one factor contributing to influenza's antigenic evolution.DOI:
http://dx.doi.org/10.7554/eLife.03300.001
“…A number of techniques have recently been described to create all amino-acid point mutants of a gene in the context of a plasmid [28][29][30]. The last few years have also seen the description of libraries of replication-competent virus mutants generated by adapting plasmid-based viral reversegenetics systems to accommodate libraries of mutagenized plasmids [31][32][33][34][35]. We utilized virus libraries created by melding these two techniques to create influenza viruses carrying all HA amino-acid point mutations compatible with viral replication [31,32].…”
Section: Resultsmentioning
confidence: 99%
“…PCR amplification of HA cDNA and Illumina sequencing library preparation was then carried out using a previously described barcoded subamplicon sequencing protocol [32], which was in turn inspired by the approach of Wu and coworkers [33]. The only change made to the previous protocol [32] was that in order to more effectively spread sequencing depth across samples based on the expected diversity of mutations in each sample, the number of uniquely-barcoded single stranded variants used as template for round 2 PCR was 5 × 10 5 to 7 × 10 5 for the noantibody control samples, and 1.5 × 10 5 for the antibody-neutralized samples.…”
Section: Deep Sequencing and Quantification Of Mutation Frequenciesmentioning
Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a high-throughput approach to quantify the selection that monoclonal antibodies exert on all single amino-acid mutations to a viral protein. This approach, mutational antigenic profiling, involves creating all replication-competent protein variants of a virus, selecting with antibody, and using deep sequencing to identify enriched mutations. We use mutational antigenic profiling to comprehensively identify mutations that enable influenza virus to escape four monoclonal antibodies targeting hemagglutinin, and validate key findings with neutralization assays. We find remarkable mutation-level idiosyncrasy in antibody escape: for instance, at a single residue targeted by two antibodies, some mutations escape both antibodies while other mutations escape only one or the other. Because mutational antigenic profiling rapidly maps all mutations selected by an antibody, it is useful for elucidating immune specificities and interpreting the antigenic consequences of viral genetic variation.
“…Transposon-based mutagenesis is a valuable and cost-effective tool that can identify critical segments of uncharacterized proteins, guiding further functional analysis (22,36,37). We acknowledge that 15-nt insertions are large enough to potentially cause significant changes to the structure or stability of the protein, which could impact our assessment of the function of that specific targeted region.…”
Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis.
IMPORTANCEThe RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.
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