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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

The sites with the greatest effect on MxA sensitivity in our experiments.(A) Differential selection from MxA for the 29 sites where total differential selection exceeds the maximum in the control selection. The logo stacks have the same meaning as in Fig 1B. The sites are ordered by total differential selection. Above the logo stacks are the avian and human consensus residues for each site. The two rows of boxes are color coded according to the percent of avian (top row) or human (bottom row) NP sequences that match the avian consensus at that site. The colors range from blue (100% match to avian consensus) to red (0% match to avian consensus). (B) The 29 sites mapped onto NP’s structure (PDB 3ZDP) as red spheres. Most sites strongly affecting MxA sensitivity are at the base of the body domain, in solvent-exposed loops, or near the N-terminus. The N-terminus and a loop (residues 392 to 407) are unresolved in the structure and so are modeled with a gray dashed line. The sites previously identified [17] as being responsible for the MxA resistance of the 1918 virus are shown as blue spheres. Site 283 is in both the 1918-resistance set and our set of 29 sites, and so is shown in purple.
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ppat.1006288.g004: The sites with the greatest effect on MxA sensitivity in our experiments.(A) Differential selection from MxA for the 29 sites where total differential selection exceeds the maximum in the control selection. The logo stacks have the same meaning as in Fig 1B. The sites are ordered by total differential selection. Above the logo stacks are the avian and human consensus residues for each site. The two rows of boxes are color coded according to the percent of avian (top row) or human (bottom row) NP sequences that match the avian consensus at that site. The colors range from blue (100% match to avian consensus) to red (0% match to avian consensus). (B) The 29 sites mapped onto NP’s structure (PDB 3ZDP) as red spheres. Most sites strongly affecting MxA sensitivity are at the base of the body domain, in solvent-exposed loops, or near the N-terminus. The N-terminus and a loop (residues 392 to 407) are unresolved in the structure and so are modeled with a gray dashed line. The sites previously identified [17] as being responsible for the MxA resistance of the 1918 virus are shown as blue spheres. Site 283 is in both the 1918-resistance set and our set of 29 sites, and so is shown in purple.

Mentions: We next sought to identify the NP sites that most affected MxA resistance. There were 29 sites where the differential selection from MxA exceeded the background noise in the control selection. Fig 4A shows these 29 sites ranked by their differential selection values. Site 283, which is one of the sites most responsible for the MxA resistance of the 1918 pandemic virus [17], ranks second in our data (Fig 4A). But most sites predicted by our deep mutational scan to have the largest effects on MxA resistance have not previously been described as impacting this phenotype. Interestingly, at all these sites, the greatest differential selection is from mutations away from the wildtype amino acid that increase MxA sensitivity.


Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA
The sites with the greatest effect on MxA sensitivity in our experiments.(A) Differential selection from MxA for the 29 sites where total differential selection exceeds the maximum in the control selection. The logo stacks have the same meaning as in Fig 1B. The sites are ordered by total differential selection. Above the logo stacks are the avian and human consensus residues for each site. The two rows of boxes are color coded according to the percent of avian (top row) or human (bottom row) NP sequences that match the avian consensus at that site. The colors range from blue (100% match to avian consensus) to red (0% match to avian consensus). (B) The 29 sites mapped onto NP’s structure (PDB 3ZDP) as red spheres. Most sites strongly affecting MxA sensitivity are at the base of the body domain, in solvent-exposed loops, or near the N-terminus. The N-terminus and a loop (residues 392 to 407) are unresolved in the structure and so are modeled with a gray dashed line. The sites previously identified [17] as being responsible for the MxA resistance of the 1918 virus are shown as blue spheres. Site 283 is in both the 1918-resistance set and our set of 29 sites, and so is shown in purple.
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Related In: Results  -  Collection

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ppat.1006288.g004: The sites with the greatest effect on MxA sensitivity in our experiments.(A) Differential selection from MxA for the 29 sites where total differential selection exceeds the maximum in the control selection. The logo stacks have the same meaning as in Fig 1B. The sites are ordered by total differential selection. Above the logo stacks are the avian and human consensus residues for each site. The two rows of boxes are color coded according to the percent of avian (top row) or human (bottom row) NP sequences that match the avian consensus at that site. The colors range from blue (100% match to avian consensus) to red (0% match to avian consensus). (B) The 29 sites mapped onto NP’s structure (PDB 3ZDP) as red spheres. Most sites strongly affecting MxA sensitivity are at the base of the body domain, in solvent-exposed loops, or near the N-terminus. The N-terminus and a loop (residues 392 to 407) are unresolved in the structure and so are modeled with a gray dashed line. The sites previously identified [17] as being responsible for the MxA resistance of the 1918 virus are shown as blue spheres. Site 283 is in both the 1918-resistance set and our set of 29 sites, and so is shown in purple.
Mentions: We next sought to identify the NP sites that most affected MxA resistance. There were 29 sites where the differential selection from MxA exceeded the background noise in the control selection. Fig 4A shows these 29 sites ranked by their differential selection values. Site 283, which is one of the sites most responsible for the MxA resistance of the 1918 pandemic virus [17], ranks second in our data (Fig 4A). But most sites predicted by our deep mutational scan to have the largest effects on MxA resistance have not previously been described as impacting this phenotype. Interestingly, at all these sites, the greatest differential selection is from mutations away from the wildtype amino acid that 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