Deep Mutational Scanning Identifies Sites in Influenza Nucleoprotein That Affect Viral Inhibition by MxA

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. Author summaryDuring 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 the sensitivity of influenza virus to MxA. While a few of these sites are known to have fixed mutations during past adaptations of influenza virus 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 Funding: This work was supported by grant R01GM102198 from the NIH/NIGMS to JDB; grant 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.

Archival influenza virus genomes from Europe reveal genomic and phenotypic variability during the 1918 pandemic

“…The availability of genome-wide information about two first wave strains also allowed us to identify two other aa changes that potentially mark a difference between first and second wave strains. We detected these variations at sites of the NP known to influence host range and susceptibility/resistance to the interferon-induced MxA antiviral protein (20,23,24): first wave strains BE-572 and BE-576 carried avian-like residues at position 16 (G) and 283 (L), whereas the fall strains and MU-162 carried D16 and P283 found in most human H1N1 strains with the exception of the 2009 pandemic H1N1 virus (see references (23)(24)(25)(26) and Fig. S12).…”

“…S12). Position 283 is located in the body of the NP and presence of a proline at this site confers resistance to the human MxA protein, to which D16 also contributes, albeit to a lower extent (24,26). In addition to supporting the hypothesis of an avian origin of this gene, these mutations may represent hallmarks of early adaptation to humans: during the first months of the pandemic, 1918 influenza viruses may have evolved a better capacity to evade the innate interferon response, which is an important aspect of influenza virus pathogenicity.…”