A CHARACTERIZATION OF BACULOVIRUS EARLY GENE EXPRESSION

描述（由申请方提供）：杆状病毒是大型DNA病毒，其特征在于在宿主昆虫中大量繁殖。由于其独特的性质，这些病原体被用作外源基因表达的载体、有效的生物杀虫剂和基因转移载体。为了确保它们的复制成功，杆状病毒使用新的机制，加速病毒基因表达和抑制宿主防御，包括细胞凋亡引起的细胞死亡。本项目的长期目标是通过确定苜蓿银纹夜蛾核型多角体病毒（Autographa californica nucleopolyhedrovirus，AcMNPV）调控基因表达和调节宿主细胞凋亡反应的机制来研究杆状病毒与宿主细胞之间的分子相互作用。在一个综合的系列实验，使用生化，遗传和细胞生物学的方法，早期AcMNPV转录调节因子加速复制的分子机制将进行调查。增强子介导的即时早期转录激活IE1的机制将通过使用新的功能丧失突变和显性抑制剂来确定。这些显性抑制剂将与干扰RNA（RNAi）结合使用，以确定IE1在感染过程中的作用，包括诱导细胞凋亡。AcMNPV感染的昆虫细胞将被用作一个强大而方便的系统来定义DNA病毒触发细胞凋亡的分子信号。利用AcMNPV在培养的果蝇细胞中诱导广泛凋亡的发现，我们使用显性抑制剂RNAi和杆状病毒凋亡抑制因子（p35，p49和iap）来研究该模型生物中的病毒和细胞凋亡因子，其中许多细胞死亡成分是已知的。这些研究将补充的分子信号（病毒和主机）的表征负责诺达病毒诱导的果蝇细胞凋亡。总的来说，这些研究有望为高度保守的细胞凋亡途径提供重要的见解，这些途径对人类病毒的致病性有重要贡献。此外，杆状病毒调节早期基因转录的分子机制与人类疾病的昆虫载体直接相关。

DESCRIPTION (provided by applicant): The baculoviruses are large DNA viruses distinguished by their prolific multiplication in host insects. Due to their unique properties, these pathogens are used as vectors for foreign gene expression, potent biological insecticides, and gene transfer vehicles. To ensure their replicative success, baculoviruses use novel mechanisms that expedite viral gene expression and suppress host defenses, including cell death by apoptosis. It is the long term goal of this project to investigate the molecular interactions between baculoviruses and their host cell by defining the mechanisms by which Autographa californica nucleopolyhedrovirus (AcMNPV) regulates gene expression and modulates the host apoptotic response. In an integrated series of experiments that use biochemical, genetic, and cell biology approaches, the molecular mechanisms by which early AcMNPV transcriptional regulators accelerate replication will be investigated. The mechanism of enhancer-mediated transcriptional activation by the immediate early transregulator IE1 will be determined by using new loss-of-function mutations and dominant inhibitors. These dominant inhibitors will be used in combination with interfering RNA (RNAi) to define the roles of IE1 during infection, including the induction of apoptosis. AcMNPV-infected insect cells will be used as a powerful yet convenient system to define the molecular signals by which DNA viruses trigger apoptosis. Capitalizing on the finding that AcMNPV induces widespread apoptosis in cultured Drosophila melanogaster cells, we use dominant inhibitors, RNAi, and the baculovirus apoptotic suppressors (p35, p49, and iap) to investigate the viral and cellular apoptotic factors in this model organism for which many of the cell death components are known. These studies will be complemented by a characterization of the molecular signals (viral and host) responsible for nodavirus-induced apoptosis in Drosophila. Collectively, these studies are expected to provide important insight into the highly conserved pathways of apoptosis, which contribute significantly to the pathogenicity of human viruses. In addition, the molecular mechanisms by which baculoviruses regulate early gene transcription are directly relevant to insect vectors of human disease.