Limits...
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.


Mutations to site 51 in NP increase MxA sensitivity as measured by polymerase activity.(A) To measure polymerase activity, we transfected unmodified MDCK-SIAT1 cells not expressing MxA with plasmids for NP and the other polymerase-complex proteins as well as a GFP reporter viral RNA. The plot shows the levels of the GFP reporter for each mutant NP relative to wildtype NP, which is set to 100%. (B) To measure the change in MxA sensitivity for each mutation, we transfected MxA-expressing and non-expressing cells with the same plasmids as in (A). For each mutant, the plot shows the levels of the GFP reporter in MxA-expressing cells normalized by the same mutant’s activity in non-expressing cells. To determine whether polymerase activity or relative activity of mutant NPs differed significantly from wildtype NP, we computed P-values using the Student’s t-test. For all mutant NPs other than D51Dsyn, the differences were significant with P<0.05 for polymerase activity and P<0.01 for relative activity.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383324&req=5

ppat.1006288.g005: Mutations to site 51 in NP increase MxA sensitivity as measured by polymerase activity.(A) To measure polymerase activity, we transfected unmodified MDCK-SIAT1 cells not expressing MxA with plasmids for NP and the other polymerase-complex proteins as well as a GFP reporter viral RNA. The plot shows the levels of the GFP reporter for each mutant NP relative to wildtype NP, which is set to 100%. (B) To measure the change in MxA sensitivity for each mutation, we transfected MxA-expressing and non-expressing cells with the same plasmids as in (A). For each mutant, the plot shows the levels of the GFP reporter in MxA-expressing cells normalized by the same mutant’s activity in non-expressing cells. To determine whether polymerase activity or relative activity of mutant NPs differed significantly from wildtype NP, we computed P-values using the Student’s t-test. For all mutant NPs other than D51Dsyn, the differences were significant with P<0.05 for polymerase activity and P<0.01 for relative activity.

Mentions: To validate the finding of our high-throughput experiments that site 51 is a major determinant of MxA sensitivity, we engineered variants of the Aichi/1968 NP carrying a variety of mutations at this site. We selected five amino-acid mutations that our high-throughput data suggest should reduce MxA resistance by varying degrees (S5 Fig). As a control, we also designed a synonymous mutation at site 51 (D51Dsyn) that was not expected to affect MxA sensitivity. To test these mutations, we measured the effect of each mutation on polymerase activity in the presence and absence of MxA. Active influenza polymerase can be reconstituted in cells, and this polymerase activity is sensitive to inhibition of NP by MxA [9]. We expected that polymerase activity would be more inhibited for mutant NPs that had increased MxA sensitivity. In the absence of MxA, the D51Dsyn mutation had similar polymerase activity to the wildtype NP while all five amino-acid mutations modestly decreased polymerase activity (Fig 5A). We compared these activities in the absence of MxA to those measured in cells expressing MxA, and quantified the effect of each mutation on MxA resistance by dividing its activity in the presence of MxA by its activity in the absence of MxA. The wildtype NP and the D51Dsyn mutant were slightly inhibited by MxA, with activity decreasing to ∼80% of its original value (Fig 5B). As predicted by our high-throughput deep mutational scanning, all five amino-acid mutants at site 51 were more strongly inhibited, with activity decreasing to between 24% and 54% of its original value (Fig 5B). This result indicates that multiple different mutations away from D at site 51 substantially increase MxA sensitivity as measured by polymerase activity.


Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA
Mutations to site 51 in NP increase MxA sensitivity as measured by polymerase activity.(A) To measure polymerase activity, we transfected unmodified MDCK-SIAT1 cells not expressing MxA with plasmids for NP and the other polymerase-complex proteins as well as a GFP reporter viral RNA. The plot shows the levels of the GFP reporter for each mutant NP relative to wildtype NP, which is set to 100%. (B) To measure the change in MxA sensitivity for each mutation, we transfected MxA-expressing and non-expressing cells with the same plasmids as in (A). For each mutant, the plot shows the levels of the GFP reporter in MxA-expressing cells normalized by the same mutant’s activity in non-expressing cells. To determine whether polymerase activity or relative activity of mutant NPs differed significantly from wildtype NP, we computed P-values using the Student’s t-test. For all mutant NPs other than D51Dsyn, the differences were significant with P<0.05 for polymerase activity and P<0.01 for relative activity.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1006288.g005: Mutations to site 51 in NP increase MxA sensitivity as measured by polymerase activity.(A) To measure polymerase activity, we transfected unmodified MDCK-SIAT1 cells not expressing MxA with plasmids for NP and the other polymerase-complex proteins as well as a GFP reporter viral RNA. The plot shows the levels of the GFP reporter for each mutant NP relative to wildtype NP, which is set to 100%. (B) To measure the change in MxA sensitivity for each mutation, we transfected MxA-expressing and non-expressing cells with the same plasmids as in (A). For each mutant, the plot shows the levels of the GFP reporter in MxA-expressing cells normalized by the same mutant’s activity in non-expressing cells. To determine whether polymerase activity or relative activity of mutant NPs differed significantly from wildtype NP, we computed P-values using the Student’s t-test. For all mutant NPs other than D51Dsyn, the differences were significant with P<0.05 for polymerase activity and P<0.01 for relative activity.
Mentions: To validate the finding of our high-throughput experiments that site 51 is a major determinant of MxA sensitivity, we engineered variants of the Aichi/1968 NP carrying a variety of mutations at this site. We selected five amino-acid mutations that our high-throughput data suggest should reduce MxA resistance by varying degrees (S5 Fig). As a control, we also designed a synonymous mutation at site 51 (D51Dsyn) that was not expected to affect MxA sensitivity. To test these mutations, we measured the effect of each mutation on polymerase activity in the presence and absence of MxA. Active influenza polymerase can be reconstituted in cells, and this polymerase activity is sensitive to inhibition of NP by MxA [9]. We expected that polymerase activity would be more inhibited for mutant NPs that had increased MxA sensitivity. In the absence of MxA, the D51Dsyn mutation had similar polymerase activity to the wildtype NP while all five amino-acid mutations modestly decreased polymerase activity (Fig 5A). We compared these activities in the absence of MxA to those measured in cells expressing MxA, and quantified the effect of each mutation on MxA resistance by dividing its activity in the presence of MxA by its activity in the absence of MxA. The wildtype NP and the D51Dsyn mutant were slightly inhibited by MxA, with activity decreasing to ∼80% of its original value (Fig 5B). As predicted by our high-throughput deep mutational scanning, all five amino-acid mutants at site 51 were more strongly inhibited, with activity decreasing to between 24% and 54% of its original value (Fig 5B). This result indicates that multiple different mutations away from D at site 51 substantially increase MxA sensitivity as measured by polymerase activity.

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.