FUNCTIONAL ANALYSIS OF DENGUE VIRUS ANTIGENS NS3 AND NS5

登革热病毒是黄病毒科的成员，被认为是 蚊媒病毒性疾病的病原体影响约1亿人 每年都有人。在这些案件中，超过25万起涉及生命危险 登革热出血热/登革休克综合征。这样做的长期目标是 实验室一直在剖析登革热涉及的生化机制 病毒在宿主体内复制。这项建议的具体目标是 定义病毒复制过程中的特定分子事件。一种体外试验 模板依赖病毒RNA复制酶检测与登革热感染性克隆 因为体外分析将被用于实现这些目标，通过实现 具体目标如下：1.病毒RNA的保守区 正链和负链rna合成都将使用野生型进行分析。 以及病毒复制检测中的突变亚基因组RNA模板。这个 亚基因组RNA将使用RNA探测方法进行分析。PI将分析 特定突变对利用病毒复制酶合成RNA的影响 以及体内的生长表型和复制效率 登革热感染性克隆。2.病毒复制酶复合体将从 表达野生型和突变型登革热病毒的重组痘苗病毒 蛋白质前体，在受感染的细胞中进行加工和组装 在活体内。研究在没有病毒粒子的情况下复制的机制 组件，一个亚基因组登革热病毒复制子，表达一个容易量化的 将构建报告基因产品。3.将检验两个假设， NS3定点突变体在病毒复制酶体外检测中的应用 野生型NS3反式互补突变基因组RNA的体内检测。 首先，具有NTPase和RNA解旋酶活性的NS3的作用是必需的 用于体外3‘端的RNA合成。第二，NS3的一个新的主题， Leu-Lys-Pro-Arg是NS3的5‘RNA三磷酸酶活性所必需的。这 许多病毒的5‘端封端的第一步需要酶的活性 RNA。PI提出，RNA的复制需要5‘端封顶。4. NS3/NS5的相互作用受磷酸化和亚细胞定位的调节 NS5。PI建议检验核NS5调节 细胞基因的表达(S)对病毒感染的反应。

Dengue viruses, members of Flaviviridae, are recognized as the causative agent of mosquito borne viral diseases affecting about 100 million people annually. Of these, more than 250,000 cases involve life threatening dengue hemorrhagic fever / dengue shock syndrome. The long term goal of this laboratory has been to dissect the biochemical mechanisms involved in dengue viral replication in the host. The specific objectives of this proposal are to define the specific molecular events during viral replication. An in vitro template dependent viral RNA replicase assay and the dengue infectious clone for in vitro analysis will be used to achieve these objectives, by fulfilling the following specific aims: 1. Conserved regions of the viral RNA required for both minus and plus strand RNA synthesis will be analyzed using the wild type and mutant subgenomic RNA templates in the viral replication assays. The subgenomic RNAs will be analyzed using RNA probing methods. The PI will analyze the effect of specific mutations on RNA synthesis using the viral replicase in vitro, and the growth phenotypes and replication efficiencies in vivo using the dengue infectious clone. 2. Viral replicase complexes will be isolated from recombinant vaccinia viruses expressing the wild type and mutant dengue viral protein precursor, which undergo processing and assembly in the infected cell in vivo. To study the mechanism of replication in the absence of virion assembly, a subgenomic dengue virus replicon expressing a readily quantifiable reporter gene product will be constructed. 3. Two hypotheses will be tested, using site directed mutants of NS3 in the in vitro viral replicase assay and the in vivo assay of mutant genomic RNAs trans-complemented by wild type NS3. First, the role of NS3, having NTPase and RNA helicase activities, is required for RNA synthesis at the 3' end in vitro. Second, a novel motif of NS3, Leu-Lys-Pro-Arg, is required for the 5'RNA triphosphatase activity of NS3. This enzyme activity is required in the first step for 5' capping of many viral RNAs. The PI propose that 5' capping is required for replication of RNA. 4. NS3/NS5 interaction is regulated by phosphorylation and subcellular location of NS5. The PI proposes to test the hypothesis that the nuclear NS5 regulates the expression of cellular gene(s) in response to viral infection.