Functional analysis of flavivirus genetic resistance

描述(申请人提供)：黄病毒属的成员，如西尼罗河病毒、日本脑炎、壁虱传播的脑炎、黄热病和登革热病毒，是人类病原体，在世界上不断扩大的地理区域造成显著的发病率和死亡率。目前，针对黄病毒的疫苗很少，也没有有效的抗病毒疗法。20世纪20年代在小鼠中首次发现的Flv基因的孟德尔显性等位基因S降低了迄今检测到的黄病毒属所有成员的复制效率，并赋予其对黄病毒诱导的疾病的抵抗力。耐药小鼠即使在脑内注射黄病毒剂量后也没有表现出任何症状，该剂量可杀死100%的同种易感小鼠。 病毒的附着和进入不会被阻止，但在耐药细胞中，细胞内病毒RNA水平和细胞外病毒产量都会减少。随后，该基因被鉴定为Oas1b基因。由于存在过早终止密码子，易感小鼠表达截短的Oas1b蛋白。用靶向同源重组将敏感的Oas1b等位基因替换为Oas1b抗性等位基因，将敏感的C57BL/6小鼠转化为耐药表型。Oas1b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。Oas1b独特的黄病毒特异性抗病毒活性不依赖于典型的核糖核酸酶L途径。最近对Oas1b细胞伙伴的鉴定为理解这种新的、天然的、黄病毒特异性的抗病毒机制提供了新的方向。初步数据： Oas1b的结合伙伴之一ABCF3表明，它特异性地参与了黄病毒的抗性机制，并可能参与了典型的OAS-核糖核酸酶L途径的调控。Oas1b结合伙伴对黄病毒特异性耐药表型的功能贡献以及对其中一个伙伴对广谱抗病毒途径的贡献将在三个特定目标下进行研究。目的1：鉴定不同类型细胞培养的耐药表型，并对Oas1b蛋白参与黄病毒耐药机制进行功能分析。目的：鉴定小鼠脑内耐药表型，分析ABCF3对Oas1b介导的小鼠黄病毒特异性耐药表型的需求。目的3：功能分析ABCF3与活性2-5A合成酶Oas1a的相互作用。该项目将提供关于一种新的、自然发生的、基因控制的、黄病毒特异性的小鼠疾病抗性机制的新知识，以及关于受这种耐药机制影响的黄病毒复制周期中独特步骤的新信息。这一信息有望为泛黄病毒抗病毒疗法的开发确定新的细胞靶点。 此外，还将获得有关OAS-核糖核酸酶L途径调控的新见解。

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.