Molecular basis of high influenza A virus susceptibility in Mus musculus castaneus carrying apparently intact Mx genes

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The Mx locus greatly determines influenza virus resistance of mice. Commonly used inbred strains such as C57BL/6 which carry defective Mx1 and Mx2 alleles are highly susceptible to influenza A virus (FLUAV) and other members of the orthomyxovirus family including Thogoto virus (THOV). By contrast, rarely used wild-derived mouse strains such as A2G which possess a functional Mx1 gene are highly resistant to FLUAV and THOV. A recent study by others showed that Mus musculus castaneus-derived CAST/EiJ mice are highly susceptible to FLUAV, although they carry an apparently intact Mx1 gene coding for a protein with a single amino acid substitution (G83R). We confirmed the FLUAV susceptibility of CAST/EiJ mice and observed that these mice are highly resistant to THOV. Finally, we found an additional change (A222V) in CAST-derived Mx1 and observed that CAST/EiJ mice contain an apparently intact Mx2 gene.Our project has two main goals. The first goal is to distinguish between the possibility that the observed amino acid changes in CAST-derived Mx1 selectively abolish activity against FLUAV but not THOV and the possibility that Mx2 accounts for the THOV resistance of CAST mice. The second goal is to understand at the molecular level how the observed two amino acid changes abolish anti-influenza activity of CAST-derived Mx1. To address the first question, we will perform FLUAV and THOV polymerase reconstitution assays in the presence or absence of transiently expressed Mx1 and Mx2 from CAST mice. These studies will be complemented by experiments in which cells over-expressing CAST-derived Mx1 or Mx2 will be analyzed for acquired resistance to infection with FLUAV and THOV. To address the second question, we will perform FLUAV polymerase reconstitution assays to determine which mutation in CAST-derived Mx1 is responsible for the loss of antiviral activity. We will further determine whether the observed amino acid changes affect GTPase activity of CAST-derived Mx1. We also plan to study the functional consequences of corresponding amino acid changes in the human MxA protein the structure of which is well defined. Finally, we will determine if CAST-derived Mx1 differs from A2G-derived Mx1 with regard to interaction with FLUAV-encoded proteins such as NP or PB2 and putative cofactors such as RNA helicases UAP56 and URH49.By studying the biological properties of a naturally occurring variant of Mx1, we hope to expand our limited knowledge on how this restriction factor is able to efficiently inhibit FLUAV replication.