Slaine, P.D.; MacRae, C.; Kleer, M.; Lamoureux, E.; McAlpine, S.; Warhuus, M.; Comeau, A.M.; McCormick, C.; Hatchette, T.; Khaperskyy, D.A. Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production. Viruses2018, 10, 272.
Slaine, P.D.; MacRae, C.; Kleer, M.; Lamoureux, E.; McAlpine, S.; Warhuus, M.; Comeau, A.M.; McCormick, C.; Hatchette, T.; Khaperskyy, D.A. Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production. Viruses 2018, 10, 272.
Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Typically, mouse-adaptation studies are conducted in inbred laboratory strains BALB/c and C57BL/6, which have defects in the antiviral Mx1 gene that results in increased susceptibility to infection and disease severity. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering these three adaptive PB1 and PA mutations into the parental CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions, and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.
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