Nürnberger Cindy, Zimmermann Vanessa, Gerhardt Melanie, Staeheli Peter
Institute of Virology, University Medical Center Freiburg, Freiburg, Germany.
Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany.
J Virol. 2016 Nov 14;90(23):10682-10692. doi: 10.1128/JVI.01213-16. Print 2016 Dec 1.
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 interferon-treated 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.
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.
A2G小鼠品系中受干扰素调节的Mx1基因赋予了对甲型流感病毒和托戈托病毒的高度抗性。大多数其他实验室近交小鼠品系携带截短的无功能Mx1等位基因，因此表现出高病毒易感性。有趣的是，源自野生小家鼠栗色亚种的CAST/EiJ小鼠拥有一个看似完整的Mx1基因，但对甲型流感病毒攻击高度敏感。为了确定CAST衍生的Mx1等位基因增强的甲型流感病毒易感性是否归因于其内在降低的抗病毒活性，我们构建了一个携带CAST/EiJ小鼠Mx基因座的近交C57BL/6J小鼠品系。该品系的成年动物对甲型流感病毒攻击的易感性几乎与缺乏功能性Mx1等位基因的标准C57BL/6J小鼠一样，但对托戈托病毒表现出更明显的抗性。测序显示，CAST衍生的MX1在GTPase结构域与A2G衍生的MX1有两个氨基酸差异（G83R和A222V）。特别是A222V突变降低了纯化的MX1的GTPase活性，并在甲型流感病毒聚合酶活性测定中减弱了MX1的抑制作用。此外，携带CAST Mx1等位基因的干扰素处理小鼠器官中的MX1蛋白比携带A2G Mx1等位基因的小鼠器官中的MX1蛋白含量显著更低。我们发现，尽管Mx1 mRNA水平未改变，但CAST特异性突变降低了MX1蛋白的代谢稳定性。因此，CAST/EiJ小鼠增强的甲型流感病毒易感性可以通过MX1限制因子中的微小改变来解释，这些改变对其酶活性产生负面影响并缩短其半衰期。
尽管原型人类MXA蛋白的晶体结构已知，但特定蛋白结构域对抗病毒活性的重要性仍未完全了解。新的见解可能来自研究具有改变的抗病毒活性的天然存在的MX蛋白变体。在这里，我们在GTPase结构域中鉴定出两个看似微小的氨基酸变化，这些变化对小鼠MX1的酶活性和代谢稳定性产生负面影响，从而显著降低了相应小鼠近交品系的甲型流感病毒抗性。这些观察结果突出了我们目前无法预测小鼠中先前未表征的MX突变的生物学后果。由于这可能也适用于人类Mx基因中的天然发生的突变，因此有必要对任何天然MXA变体的活性改变进行仔细的实验分析，以评估此类突变对先天抗病毒免疫的可能后果。
