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Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

View Article: PubMed Central - PubMed

ABSTRACT

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.

No MeSH data available.


Related in: MedlinePlus

A deep mutational scan for NP mutations that affect MxA resistance.(A) We created a library of influenza variants carrying all viable amino-acid mutants of NP. We infected cells expressing human MxA, not expressing human MxA, or expressing inactive MxA, and used deep sequencing to quantify the enrichment or depletion of each mutation in each condition. (B) The effect of each mutation on MxA sensitivity is computed as the logarithm of its relative frequency in MxA-expressing versus non-expressing cells. These measurements are summarized in the logo plots, where letters above the black horizontal line represent amino acids that increase MxA resistance, and letters below the line represent amino acids that increase MxA sensitivity. The overall differential selection at a site is the total height of the letter stack. Letters are colored according to hydrophobicity. The cells expressing the inactive MxA provide a control to estimate experimental noise. At the example site shown (site 283), most mutations increase MxA sensitivity, and the actual differential selection in the MxA-expressing cells is much greater than the noise measured in the cells expressing inactive MxA.
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ppat.1006288.g001: A deep mutational scan for NP mutations that affect MxA resistance.(A) We created a library of influenza variants carrying all viable amino-acid mutants of NP. We infected cells expressing human MxA, not expressing human MxA, or expressing inactive MxA, and used deep sequencing to quantify the enrichment or depletion of each mutation in each condition. (B) The effect of each mutation on MxA sensitivity is computed as the logarithm of its relative frequency in MxA-expressing versus non-expressing cells. These measurements are summarized in the logo plots, where letters above the black horizontal line represent amino acids that increase MxA resistance, and letters below the line represent amino acids that increase MxA sensitivity. The overall differential selection at a site is the total height of the letter stack. Letters are colored according to hydrophobicity. The cells expressing the inactive MxA provide a control to estimate experimental noise. At the example site shown (site 283), most mutations increase MxA sensitivity, and the actual differential selection in the MxA-expressing cells is much greater than the noise measured in the cells expressing inactive MxA.

Mentions: Our goal is to understand which sites in influenza NP determine its sensitivity to MxA. We can do this by experimentally quantifying how MxA sensitivity is affected by all amino-acid mutations to NP that support viral replication. Systematic measurements of this type can be made using the deep mutational scanning approach outlined in Fig 1. This approach involves creating influenza viruses that carry a diverse set of NP mutations, growing these viruses in cells that do or do not express human MxA, and then using deep sequencing to identify mutations that are enriched or depleted in one condition versus the other. Mutations that are enriched in MxA-expressing cells relative to control cells increase MxA resistance, whereas mutations that are relatively depleted in MxA-expressing cells increase MxA sensitivity.


Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA
A deep mutational scan for NP mutations that affect MxA resistance.(A) We created a library of influenza variants carrying all viable amino-acid mutants of NP. We infected cells expressing human MxA, not expressing human MxA, or expressing inactive MxA, and used deep sequencing to quantify the enrichment or depletion of each mutation in each condition. (B) The effect of each mutation on MxA sensitivity is computed as the logarithm of its relative frequency in MxA-expressing versus non-expressing cells. These measurements are summarized in the logo plots, where letters above the black horizontal line represent amino acids that increase MxA resistance, and letters below the line represent amino acids that increase MxA sensitivity. The overall differential selection at a site is the total height of the letter stack. Letters are colored according to hydrophobicity. The cells expressing the inactive MxA provide a control to estimate experimental noise. At the example site shown (site 283), most mutations increase MxA sensitivity, and the actual differential selection in the MxA-expressing cells is much greater than the noise measured in the cells expressing inactive MxA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383324&req=5

ppat.1006288.g001: A deep mutational scan for NP mutations that affect MxA resistance.(A) We created a library of influenza variants carrying all viable amino-acid mutants of NP. We infected cells expressing human MxA, not expressing human MxA, or expressing inactive MxA, and used deep sequencing to quantify the enrichment or depletion of each mutation in each condition. (B) The effect of each mutation on MxA sensitivity is computed as the logarithm of its relative frequency in MxA-expressing versus non-expressing cells. These measurements are summarized in the logo plots, where letters above the black horizontal line represent amino acids that increase MxA resistance, and letters below the line represent amino acids that increase MxA sensitivity. The overall differential selection at a site is the total height of the letter stack. Letters are colored according to hydrophobicity. The cells expressing the inactive MxA provide a control to estimate experimental noise. At the example site shown (site 283), most mutations increase MxA sensitivity, and the actual differential selection in the MxA-expressing cells is much greater than the noise measured in the cells expressing inactive MxA.
Mentions: Our goal is to understand which sites in influenza NP determine its sensitivity to MxA. We can do this by experimentally quantifying how MxA sensitivity is affected by all amino-acid mutations to NP that support viral replication. Systematic measurements of this type can be made using the deep mutational scanning approach outlined in Fig 1. This approach involves creating influenza viruses that carry a diverse set of NP mutations, growing these viruses in cells that do or do not express human MxA, and then using deep sequencing to identify mutations that are enriched or depleted in one condition versus the other. Mutations that are enriched in MxA-expressing cells relative to control cells increase MxA resistance, whereas mutations that are relatively depleted in MxA-expressing cells increase MxA sensitivity.

View Article: PubMed Central - PubMed

ABSTRACT

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.

No MeSH data available.


Related in: MedlinePlus