FUNCTIONAL ANALYSIS OF DENGUE VIRUS ANTIGENS NS3 AND NS5

登革病毒，黄病毒科的成员，被认为是 蚊子传播病毒性疾病的病原体，影响约一亿人 人每年。其中，超过25万例涉及危及生命 登革出血热/登革休克综合征。长期目标是 一个实验室一直在研究登革热的生化机制 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。体外 模板依赖性病毒RNA复制酶测定和登革感染性克隆 用于体外分析，将通过满足以下条件来实现这些目标： 具体目标如下：1.病毒RNA的保守区域， 负链和正链RNA合成都将使用野生型 和突变亚基因组RNA模板。的 将使用RNA探测方法分析亚基因组RNA。PI将分析 特定突变对使用病毒复制酶的RNA合成的影响， 体外，和生长表型和复制效率在体内使用的 登革热感染性克隆。2.病毒复制酶复合物将从 表达野生型和突变型登革病毒的重组牛痘病毒 蛋白质前体，在感染细胞中进行加工和组装 in vivo.研究病毒粒子缺失时的复制机制 组装，亚基因组登革病毒复制子表达易于定量的 构建报告基因产物。3.将检验两个假设， 在体外病毒复制酶测定中使用NS 3的定点突变体， 通过野生型NS 3反式互补的突变基因组RNA的体内测定。 首先，需要具有NTR和RNA解旋酶活性的NS 3的作用 用于体外3'端的RNA合成。第二，NS 3的一个新的母题， Leu-Lys-Pro-Arg是NS 3的5 'RNA三磷酸酶活性所必需的。这 酶活性在许多病毒的5 ′加帽的第一步中是必需的 RNA。PI提出5'加帽是RNA复制所必需的。4. NS 3/NS 5的相互作用是通过磷酸化和亚细胞定位来调节的。 NS5。PI建议测试核NS 5调节细胞核内蛋白质的假说。 响应病毒感染的细胞基因的表达。

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.