BACULOVIRUS EARLY GENE EXPRESSION

杆状病毒是一个致病的大DNA病毒家族 到昆虫。最近用新毒素改造这些病毒 作为生物杀虫剂的改良基因引起了新的关注 关于宿主范围和对非靶生物的安全性， 包括人类 对杆状病毒安全性的正确评价要求 增加对控制宿主的分子机制的理解 范围和病毒复制。 本提案的长期目标是 研究杆状病毒最早期基因的功能和调控 确定宿主-病毒相互作用和病毒复制的机制 战略布局 我们将主要关注早期35 K蛋白（p35）基因 由原型杆状病毒，苜蓿银纹夜蛾 （多衣壳）核型多角体病毒（AcMNPV）。p35是关键 病毒以细胞特异性方式复制。 它还可以防止过早 病毒诱导的程序性细胞死亡导致的宿主细胞裂解 （凋亡）。 细胞凋亡是一个内在的、信号诱导的过程， 细胞自毁;它被用来控制细胞数量。 制止 凋亡导致异常的细胞存活，因此有助于 肿瘤发生 所涉及的机制尚不清楚。 AcMNPV是第一个 已知在非免疫来源的细胞中诱导细胞凋亡的病毒， 首先同时编码抑制该宿主的基因（p35 反应 p35突变体产生的容易程度和 培养应答细胞的相对便利性提供了新的 研究程序性细胞死亡调节的机会， 研究细胞凋亡作为抗病毒防御的可能作用 战略 在这个建议中，我们使用了一个组合的网站导向 突变和新的重组病毒，以检查机制， 其中p35阻断AcMNPV感染的细胞的凋亡。 遗传和 将采用生物化学方法来探索诱导细胞凋亡 及其对复制的影响，包括病毒DNA片段化和基因 表情 我们还将使用p35基因作为敏感的报告基因， 早期复制事件，以便继续定义顺式作用 序列和反式作用因子（宿主和病毒）参与 杆状病毒转录调控 我们在这里重点关注监管 启动子上游激活区中的基序，5' 非编码RNA前导序列和病毒增强子，每一个已知的影响早期 转录。 最后，已经证明p35基因可以被 作为一个强大的选择标记（杆状病毒的第一个），我们 开发一种新的，基于转座子的随机诱变系统， AcMNPV基因组是继续研究病毒基因的关键 组织和功能。 总之，这些研究预计将 对真核基因的复杂机制产生重要的洞察力 调节以及诱导和抑制程序化细胞 与控制细胞增殖有关的死亡。

The baculoviruses are a family of large DNA viruses that are pathogenic to insects. The recent engineering of these viruses with novel toxin genes for improved properties as bio-pesticides has raised new concerns about host range and safety with respect to non-target organisms, including humans. Proper evaluation of baculovirus safety has demanded an increased understanding of the molecular mechanisms controlling host range and virus replication. The long term goal of this proposal is to examine the function and regulation of the earliest of baculovirus genes to define the mechanisms of host-virus interactions and virus replication strategies. We will focus primarily on the early 35K protein (p35) gene encoded by the prototype baculovirus, Autographa californica (multicapsid) nuclear polyhedrosis virus (AcMNPV). p35 is critical for virus replication in a cell-specific manner. It also prevents premature host cell lysis that is the result of virus-induced programmed cell death (apoptosis). Apoptosis is a built-in, signal-induced process by which a cell self-destructs; it is used to control cell numbers. Suppression of apoptosis results in aberrant cell survival and therefore contributes to oncogenesis. The mechanisms involved are unknown. AcMNPV is the first virus known to induce apoptosis in cells of non-immune origin and the first to simultaneously encode a gene (p35) for suppression of this host response. The ease with which p35 mutants can be generated and the relative convenience of culturing the responsive cells provide new opportunities to investigate the regulation of programmed cell death and to examine the possible role of apoptosis as an anti-virus defense strategy. In this proposal, we use a combination of site-directed mutagenesis and novel recombinant viruses to examine the mechanism by which p35 blocks apoptosis of AcMNPV-infected cells. Genetic and biochemical approaches will be used to explore the induction of apoptosis and its effect on replication, including virus DNA fragmentation and gene expression. We will also use the p35 gene as a sensitive reporter of early replication events in order to continue defining the cis-acting sequences and trans-acting factors (host and virus) involved in baculovirus transcriptional regulation. We focus here on the regulatory motifs in the upstream activating region of the promoter, the 5' noncoding RNA leader, and virus enhancers, each known to affect early transcription. Finally, having demonstrated that the p35 gene can be used as a powerful selectable marker (the first for baculoviruses), we develop a novel, transposon-based system for random mutagenesis of the AcMNPV genome that is critical for continued studies on virus gene organization and function. In summary, these studies are expected to yield important insight into the complex mechanisms of eukaryotic gene regulation as well as the induction and suppression of programmed cell death that is relevant to the control of cell proliferation.