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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

Validation of mutational effects on NP sensitivity.We performed competition experiments between the wildtype virus and each mutant virus in MxA-expressing and non-expressing cells. The plots show the relative frequency of each mutant virus in MxA-expressing versus non-expressing cells at 10 hours and 54 hours post-infection. This enrichment is less than one if the mutation increases MxA sensitivity, and greater than one if the mutation increases MxA resistance. Blue points correspond to mutations predicted by the deep mutational scanning to increase MxA sensitivity, red points correspond to mutations predicted to increase MxA resistance, and the black point corresponds to a synonymous mutation predicted to have no effect. In all cases, the results of these validation experiments are consistent with the deep mutational scanning results.
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ppat.1006288.g007: Validation of mutational effects on NP sensitivity.We performed competition experiments between the wildtype virus and each mutant virus in MxA-expressing and non-expressing cells. The plots show the relative frequency of each mutant virus in MxA-expressing versus non-expressing cells at 10 hours and 54 hours post-infection. This enrichment is less than one if the mutation increases MxA sensitivity, and greater than one if the mutation increases MxA resistance. Blue points correspond to mutations predicted by the deep mutational scanning to increase MxA sensitivity, red points correspond to mutations predicted to increase MxA resistance, and the black point corresponds to a synonymous mutation predicted to have no effect. In all cases, the results of these validation experiments are consistent with the deep mutational scanning results.

Mentions: All mutations clearly had the predicted effect on MxA sensitivity at the 54-hour timepoint, and at least weakly had the predicted effect at the earlier 10-hour timepoint when selection has had less time to act (Fig 7). As expected, D51N and the eight additional putative sensitizing mutations were depleted in the MxA-expressing cells relative to the control cells, with D51N having the strongest effect (Fig 7). The three putative resistance mutations were all enriched in the MxA-expressing cells relative to the control cells, validating that they do indeed increase resistance (Fig 7). The largest increase in resistance was conferred by the R102A mutation. This resistance mutation also substantially attenuates viral growth (S7 Fig), perhaps explaining why it is not fixed in the human influenza NP. However, there does not seem to be any general trend for mutations to have similar effects on viral growth and MxA resistance, as we identify attenuating mutations that increase both MxA resistance (e.g., R102A) and sensitivity (e.g., D51N), while also identifying mutations with both effects on MxA sensitivity that do not greatly affect viral growth (e.g., E294R and Q399R). As expected, the control synonymous D51Dsyn mutation had no effect on MxA sensitivity (Fig 7). These results demonstrate that our deep mutational scanning approach accurately identifies mutations that increase both sensitivity and resistance to MxA.


Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA
Validation of mutational effects on NP sensitivity.We performed competition experiments between the wildtype virus and each mutant virus in MxA-expressing and non-expressing cells. The plots show the relative frequency of each mutant virus in MxA-expressing versus non-expressing cells at 10 hours and 54 hours post-infection. This enrichment is less than one if the mutation increases MxA sensitivity, and greater than one if the mutation increases MxA resistance. Blue points correspond to mutations predicted by the deep mutational scanning to increase MxA sensitivity, red points correspond to mutations predicted to increase MxA resistance, and the black point corresponds to a synonymous mutation predicted to have no effect. In all cases, the results of these validation experiments are consistent with the deep mutational scanning results.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1006288.g007: Validation of mutational effects on NP sensitivity.We performed competition experiments between the wildtype virus and each mutant virus in MxA-expressing and non-expressing cells. The plots show the relative frequency of each mutant virus in MxA-expressing versus non-expressing cells at 10 hours and 54 hours post-infection. This enrichment is less than one if the mutation increases MxA sensitivity, and greater than one if the mutation increases MxA resistance. Blue points correspond to mutations predicted by the deep mutational scanning to increase MxA sensitivity, red points correspond to mutations predicted to increase MxA resistance, and the black point corresponds to a synonymous mutation predicted to have no effect. In all cases, the results of these validation experiments are consistent with the deep mutational scanning results.
Mentions: All mutations clearly had the predicted effect on MxA sensitivity at the 54-hour timepoint, and at least weakly had the predicted effect at the earlier 10-hour timepoint when selection has had less time to act (Fig 7). As expected, D51N and the eight additional putative sensitizing mutations were depleted in the MxA-expressing cells relative to the control cells, with D51N having the strongest effect (Fig 7). The three putative resistance mutations were all enriched in the MxA-expressing cells relative to the control cells, validating that they do indeed increase resistance (Fig 7). The largest increase in resistance was conferred by the R102A mutation. This resistance mutation also substantially attenuates viral growth (S7 Fig), perhaps explaining why it is not fixed in the human influenza NP. However, there does not seem to be any general trend for mutations to have similar effects on viral growth and MxA resistance, as we identify attenuating mutations that increase both MxA resistance (e.g., R102A) and sensitivity (e.g., D51N), while also identifying mutations with both effects on MxA sensitivity that do not greatly affect viral growth (e.g., E294R and Q399R). As expected, the control synonymous D51Dsyn mutation had no effect on MxA sensitivity (Fig 7). These results demonstrate that our deep mutational scanning approach accurately identifies mutations that increase both sensitivity and resistance to 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