Functional analysis of flavivirus genetic resistance

描述（由申请人提供）：黄病毒属的成员，如西尼罗河病毒、日本脑炎病毒、蜱传脑炎病毒、黄热病病毒和登革热病毒，是在世界不断扩大的地理区域中引起显著发病率和死亡率的人类病原体。目前，对于黄病毒，疫苗很少，并且没有可用的有效的抗病毒疗法。在20世纪20年代首次在小鼠中发现的Flv基因的孟德尔显性等位基因降低了迄今为止测试的黄病毒属所有成员的复制效率，并赋予对黄病毒诱导的疾病的抗性。耐药小鼠即使在脑内注射100%杀死同类易感小鼠的黄病毒剂量后也没有表现出任何症状。 病毒附着和进入未被阻断，但在抗性细胞中细胞内病毒RNA水平和细胞外病毒产生减少。Flv基因座随后被鉴定为Oas 1b基因。易感小鼠由于存在提前终止密码子而表达截短的Oas 1b蛋白。使用靶向同源重组将易感的C57 BL/6小鼠转化为抗性表型，通过敲入Oas 1b抗性等位基因来替换易感的Oas 1b等位基因。Oas 1b是2-5A合成酶家族的成员，但该基因的两个等位基因的产物都是失活的合成酶。Oas 1b独特的黄病毒特异性抗病毒活性不依赖于经典的RNase L途径。最近对Oas 1b细胞伴侣的鉴定为了解这种新型、天然、黄病毒特异性的抗病毒机制提供了新的方向。年的初步数据 Oas 1b结合配偶体之一ABCF 3表明其特异性参与黄病毒抗性机制，并且其也可能参与调节经典Oas-RNase L途径。Oas 1b结合伴侣对黄病毒特异性耐药表型的功能贡献，以及其中一个伴侣对广谱抗病毒途径的贡献将在三个特定目标下进行研究。目标1：在各种类型的细胞培养物中的抗性表型的表征和黄病毒抗性机制中Oas 1b伴侣蛋白参与的功能分析。目标二：小鼠脑中耐药表型的表征和小鼠中Oas 1b介导的黄病毒特异性耐药表型对ABCF 3的需求分析。目的3：ABCF 3与2-5A合成酶Oas 1a相互作用的功能分析。该项目将提供有关小鼠中一种新型的、天然存在的、遗传控制的黄病毒特异性疾病抗性机制的新知识，并揭示有关黄病毒复制周期中受这种抗性机制影响的独特步骤的新信息。这些信息有望为泛黄病毒抗病毒治疗的开发确定新的细胞靶点。 还将获得关于Oas-RNase L途径调节的新见解。

DESCRIPTION (provided by applicant): Members of the genus Flaviviruses, such as West Nile, Japanese encephalitis, tick-borne encephalitis, yellow fever, and dengue virus, are human pathogens that cause significant morbidity and mortality in ever-widening geographic areas of the world. Currently, there are few vaccines and no available efficacious antiviral therapies for flaviviruses. A Mendelian dominant allele of the Flv gene that was first discovered in mice in the 1920's reduces the replication efficiency of all members of the genus Flavivirus so far tested and confers resistance to flavivirus-induced disease. Resistant mice display no symptoms even after an intracerebral injection of a flavivirus dose that kills 100% of congenic susceptible mice. Viral attachment and entry are not blocked but intracellular viral RNA levels and extracellular virus production are reduced in resistant cells. The Flv locus was subsequently identified as the Oas1b gene. Susceptible mice express a truncated Oas1b protein due to the presence of a premature stop codon. Replacement of the susceptible Oas1b allele by knock-in of the Oas1b resistance allele using targeted homologous recombination converted susceptible C57BL/6 mice to the resistant phenotype. Oas1b is a member of the 2-5A synthetase family but the products of both alleles of this gene are inactive synthetases. The unique flavivirus-specific antiviral activity of Oas1b is not dependent on the canonical RNase L pathway. The recent identification of cell partners for Oas1b has provided new directions for gaining an understanding of this novel, natural, flavivirus-specific antiviral mechanism. Preliminary data for one of the Oas1b binding partners, ABCF3, indicate that it is specifically involved in the flavivirus-resistance mechanism and that it may also be involved in regulating the canonical Oas-RNase L pathway. Functional contributions of the Oas1b binding partners to the flavivirus-specific resistance phenotype and for one of the partners, a contribution to a broad spectrum antiviral pathway will be studied under three specific aims. Aim 1: Characterization of the resistance phenotype in various types of cell cultures and functional analyses of the involvement of Oas1b partner proteins in the flavivirus resistance mechanism. Aim 2: Characterization of the resistance phenotype in mouse brains and analysis of the requirement of ABCF3 for the Oas1b-mediated, flavivirus-specific resistance phenotype in mice. Aim 3: Functional analysis of the interaction of ABCF3 with the active 2-5A synthetase Oas1a. This project will provide new knowledge about a novel, naturally-occurring, genetically-controlled, flavivirus-specific disease resistance mechanism in mice as well as reveal new information about the unique step in the flavivirus replication cycle that is affected by this resistance mechanism. This information is expected to identify new cell targets for the development of pan-flavivirus antiviral therapeutics. New insights about the regulation of the Oas-RNase L pathway will also be obtained.