Influenza Virus Susceptibility of Wild-Derived CAST/EiJ Mice Results from Two Amino Acid Changes in the MX1 Restriction Factor
Influenza Virus Susceptibility of Wild-Derived CAST/EiJ Mice Results from Two Amino Acid Changes in the MX1 Restriction Factor
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DOI：
10.1128/jvi.01213-16
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发表时间：
2016-09
影响因子：
5.4
通讯作者：
C. Nürnberger;Vanessa Zimmermann;M. Gerhardt;P. Staeheli
中科院分区：
文献类型：
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作者：
C. Nürnberger;Vanessa Zimmermann;M. Gerhardt;P. Staeheli
A2 G小鼠品系的干扰素调节的Mx 1基因赋予对甲型流感病毒和Thogoto病毒的高度抗性。大多数其他实验室近交系小鼠品系携带截短的非功能性Mx 1等位基因，因此表现出高病毒易感性。有趣的是，来自野生小家鼠的CAST/EiJ小鼠具有看似完整的Mx 1基因，但对甲型流感病毒攻击高度敏感。为了确定增强的流感病毒易感性是否是由于CAST衍生的Mx 1等位基因的内在降低的抗病毒活性，我们产生了携带CAST/EiJ小鼠的Mx基因座的同源C57 BL/6 J小鼠系。该品系的成年动物几乎与缺乏功能性Mx 1等位基因的标准C57 BL/6 J小鼠一样易受流感病毒攻击，但对Thogoto病毒表现出明显得多的抗性。测序显示，CAST衍生的MX 1与A2 G衍生的MX 1在GT3结构域中的两个氨基酸（G83 R和A222 V）不同。特别是A222 V突变降低了纯化的MX 1的GTdR活性，并减弱了MX 1在甲型流感病毒聚合酶活性测定中的抑制作用。此外，MX 1蛋白在携带CAST Mx 1等位基因的干扰素治疗小鼠的器官中的丰度显著低于携带A2 G Mx 1等位基因的小鼠。我们发现，CAST特异性突变降低了MX 1蛋白的代谢稳定性，尽管Mx 1 mRNA水平没有变化。因此，CAST/EiJ小鼠的流感病毒易感性增强可以通过MX 1限制因子的微小改变来解释，MX 1限制因子的微小改变对其酶活性产生负面影响并缩短其半衰期。重要性尽管原型人MXA蛋白的晶体结构是已知的，但特异性蛋白质结构域对于抗病毒活性的重要性仍不完全清楚。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，我们确定了两个看似微小的氨基酸变化的GT3结构域，产生负面影响的酶活性和代谢稳定性的小鼠MX 1，从而显着降低流感病毒的耐药性相应的小鼠近交系。这些观察结果突出表明，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。由于这对于人类Mx基因中天然存在的突变也可能是正确的，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。
ABSTRACT 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. 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.