Influenza Virus Susceptibility of Wild-Derived CAST/EiJ Mice Results from Two Amino Acid Changes in the MX1 Restriction Factor

The interferon-regulated Mx1 gene of the A2G mouse strain confers a high degree of resistance against influenza A and Thogoto viruses. Most other laboratory inbred mouse strains carry truncated nonfunctional Mx1 alleles and, consequently, exhibit high virus susceptibility. Interestingly, CAST/EiJ mice, derived from wild Mus musculus castaneus, possess a seemingly intact Mx1 gene but are highly susceptible to influenza A virus challenge. To determine whether the enhanced influenza virus susceptibility is due to intrinsically reduced antiviral activity of the CAST-derived Mx1 allele, we generated a congenic C57BL/6J mouse line that carries the Mx locus of CAST/EiJ mice. Adult animals of this line were almost as susceptible to influenza virus challenge as standard C57BL/6J mice lacking functional Mx1 alleles but exhibited far more pronounced resistance to Thogoto virus. Sequencing revealed that CAST-derived MX1 differs from A2G-derived MX1 by two amino acids (G83R and A222V) in the GTPase domain. Especially the A222V mutation reduced GTPase activity of purified MX1 and diminished the inhibitory effect of MX1 in influenza A virus polymerase activity assays. Further, MX1 protein was substantially less abundant in organs of interferontreated mice carrying the CAST Mx1 allele than in those of mice carrying the A2G Mx1 allele. We found that the CAST-specific mutations reduced the metabolic stability of the MX1 protein although Mx1 mRNA levels were unchanged. Thus, the enhanced influenza virus susceptibility of CAST/EiJ mice can be explained by minor alterations in the MX1 restriction factor that negatively affect its enzymatic activity and reduce its half-life. IMPORTANCE Although the crystal structure of the prototypic human MXA protein is known, the importance of specific protein domains for antiviral activity is still incompletely understood. Novel insights might come from studying naturally occurring MX protein variants with altered antiviral activity. Here we identified two seemingly minor amino acid changes in the GTPase domain that negatively affect the enzymatic activity and metabolic stability of murine MX1 and thus dramatically reduce the influenza virus resistance of the respective mouse inbred strain. These observations highlight our current inability to predict the biological consequences of previously uncharacterized MX mutations in mice. Since this is probably also true for naturally occurring mutations in Mx genes of humans, careful experimental analysis of any natural MXA variants for altered activity is necessary in order to assess possible consequences of such mutations on innate antiviral immunity.