PT  - JOURNAL ARTICLE
AU  - Orr Ashenberg
AU  - Jai Padmakumar
AU  - Michael B. Doud
AU  - Jesse D. Bloom
TI  - Deep Mutational Scanning Identifies Sites in Influenza Nucleoprotein That Affect Viral Inhibition by MxA
AID  - 10.1101/071969
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 071969
4099  - http://biorxiv.org/content/early/2016/08/28/071969.short
4100  - http://biorxiv.org/content/early/2016/08/28/071969.full
AB  - The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza is more resistant than avian influenza 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 ≈10,000 amino-acid mutations to NP affect MxA sensitivity. 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 interactions with host factors.Author Summary During viral infection, human cells express proteins that can restrict virus replication. However, in many cases it remains unclear what determines the sensitivity of a given viral strain to a particular restriction factor. Here we use a high-throughput approach to measure how all amino-acid mutations to the nucleoprotein of influenza virus affect restriction by the human protein MxA. We find several dozen sites where mutations substantially affect influenza’s sensitivity to MxA. While a few of these sites are known to have fixed mutations during past adaptations of influenza to humans, most of the sites are broadly conserved across all influenza strains and have never previously been described as affecting MxA resistance. Our results therefore show that the known historical evolution of influenza has only involved substitutions at a small fraction of the sites where mutations can in principle affect MxA resistance. We suggest that this is because many sites are already broadly fixed at amino acids that confer high resistance.