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

Mx基因座在很大程度上决定了小鼠对流感病毒的抵抗力。通常使用的近交系菌株，如C57BL/6，携带有缺陷的Mx1和Mx2等位基因，对甲型流感病毒(FLUAV)和包括托戈托病毒(Thogoto Virus)在内的正粘病毒家族的其他成员高度敏感。相比之下，很少使用的野生小鼠品系，如拥有功能性Mx1基因的A2G，对FLUAV和THOV具有高度抵抗力。最近的一项研究表明，卡氏小鼠来源的CAST/EIJ小鼠对FLUAV高度易感，尽管它们携带一个表面上完整的Mx1基因，编码一种具有单一氨基酸取代的蛋白质(G83R)。我们证实了CAST/EIJ小鼠对FLUAV的敏感性，并观察到这些小鼠对THOV具有高度的抵抗力。最后，我们在CAST衍生的MX1中发现了一个额外的变化(A222V)，并观察到CAST/EIJ小鼠包含一个明显完整的MX2基因。我们的项目有两个主要目标。第一个目标是区分观察到的CAST来源的MX1中的氨基酸变化选择性地取消对FLUAV而不是THOV活性的可能性，以及MX2解释CAST小鼠THOV抵抗的可能性。第二个目标是在分子水平上了解观察到的两个氨基酸的变化如何取消CAST衍生的Mx1的抗流感活性。为了解决第一个问题，我们将在存在或不存在CAST小鼠瞬时表达的MX1和MX2的情况下进行FLUAV和THOV聚合酶重构分析。这些研究将得到实验的补充，在实验中，将分析过度表达CAST衍生的MX1或MX2的细胞对FLUAV和THOV感染的获得性抵抗力。为了解决第二个问题，我们将进行FLUAV聚合酶重组分析，以确定CAST衍生的Mx1中的哪个突变导致抗病毒活性的丧失。我们将进一步确定观察到的氨基酸变化是否影响CAST来源的Mx1的GTP酶活性。我们还计划研究人类MxA蛋白中相应氨基酸变化的功能后果，该蛋白的结构已经很好地定义了。最后，我们将确定CAST来源的Mx1与A2G来源的Mx1在与FLUAV编码的蛋白质(如NP或PB2)以及可能的辅助因子(如RNA解旋酶UAP56和URH49)的相互作用方面是否不同。通过研究Mx1的自然变体的生物学特性，我们希望扩大我们有限的知识，即这种限制因子如何能够有效地抑制FLUAV的复制。

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.