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The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin.

Thyagarajan B, Bloom JD - Elife (2014)

Bottom Line: We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.

ABSTRACT
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 ≈10(4) 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.

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Related in: MedlinePlus

Schematic of the deep mutational scanning experiment.The Illumina deep-sequencing samples are shown in yellow boxes (DNA, mutDNA, virus, mutvirus). Experimental steps and associated sources of mutations are shown in blue text, while sources of error during Illumina sample preparation and sequencing are shown in red text. This entire process was performed in biological triplicate.DOI:http://dx.doi.org/10.7554/eLife.03300.003
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fig1: Schematic of the deep mutational scanning experiment.The Illumina deep-sequencing samples are shown in yellow boxes (DNA, mutDNA, virus, mutvirus). Experimental steps and associated sources of mutations are shown in blue text, while sources of error during Illumina sample preparation and sequencing are shown in red text. This entire process was performed in biological triplicate.DOI:http://dx.doi.org/10.7554/eLife.03300.003

Mentions: Our strategy for deep mutational scanning (Fowler et al., 2010; Araya and Fowler, 2011) of HA is outlined in Figure 1. The wildtype WSN HA gene was mutagenized to create a diverse library of mutant HA genes. This library of mutant genes was then used to generate a pool of mutant viruses by reverse genetics (Hoffmann et al., 2000). The mutant viruses were passaged at a low multiplicity of infection to ensure a linkage between genotype and phenotype. The frequencies of mutations before and after selection for viral growth were quantified by Illumina deep sequencing of the mutant genes (the mutDNA sample in Figure 1) and the mutant viruses (the mutvirus sample in Figure 1). An identical process was performed in parallel using the unmutated wildtype HA gene to generate unmutated viruses in order to quantify the error rates associated with sequencing, reverse transcription, and virus growth (these are the DNA and virus samples in Figure 1). The entire process in Figure 1 was performed in full biological triplicate (the replicates are referred to as #1, #2, and #3). In addition, a repeat of the Illumina sample preparation and deep sequencing was performed for replicate #1 to quantify the technical variation associated with these processes.10.7554/eLife.03300.003Figure 1.Schematic of the deep mutational scanning experiment.


The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin.

Thyagarajan B, Bloom JD - Elife (2014)

Schematic of the deep mutational scanning experiment.The Illumina deep-sequencing samples are shown in yellow boxes (DNA, mutDNA, virus, mutvirus). Experimental steps and associated sources of mutations are shown in blue text, while sources of error during Illumina sample preparation and sequencing are shown in red text. This entire process was performed in biological triplicate.DOI:http://dx.doi.org/10.7554/eLife.03300.003
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109307&req=5

fig1: Schematic of the deep mutational scanning experiment.The Illumina deep-sequencing samples are shown in yellow boxes (DNA, mutDNA, virus, mutvirus). Experimental steps and associated sources of mutations are shown in blue text, while sources of error during Illumina sample preparation and sequencing are shown in red text. This entire process was performed in biological triplicate.DOI:http://dx.doi.org/10.7554/eLife.03300.003
Mentions: Our strategy for deep mutational scanning (Fowler et al., 2010; Araya and Fowler, 2011) of HA is outlined in Figure 1. The wildtype WSN HA gene was mutagenized to create a diverse library of mutant HA genes. This library of mutant genes was then used to generate a pool of mutant viruses by reverse genetics (Hoffmann et al., 2000). The mutant viruses were passaged at a low multiplicity of infection to ensure a linkage between genotype and phenotype. The frequencies of mutations before and after selection for viral growth were quantified by Illumina deep sequencing of the mutant genes (the mutDNA sample in Figure 1) and the mutant viruses (the mutvirus sample in Figure 1). An identical process was performed in parallel using the unmutated wildtype HA gene to generate unmutated viruses in order to quantify the error rates associated with sequencing, reverse transcription, and virus growth (these are the DNA and virus samples in Figure 1). The entire process in Figure 1 was performed in full biological triplicate (the replicates are referred to as #1, #2, and #3). In addition, a repeat of the Illumina sample preparation and deep sequencing was performed for replicate #1 to quantify the technical variation associated with these processes.10.7554/eLife.03300.003Figure 1.Schematic of the deep mutational scanning experiment.

Bottom Line: We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.

ABSTRACT
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 ≈10(4) 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.

Show MeSH
Related in: MedlinePlus