BACULOVIRUS EARLY GENE EXPRESSION

杆状病毒早期基因表达

• 批准号: 2063005
• 负责人: PAUL D FRIESEN
• 金额: $ 18.48万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2063005
• 关键词: Baculoviridae; apoptosis; cell growth regulation; gene expression; genetic regulation; genetic regulatory element; genetic transcription; host organism interaction; nucleic acid sequence; site directed mutagenesis; virulence; virus genetics; virus infection mechanism; virus replication

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.

杆状病毒是一类具有致病性的大型dna病毒。 敬昆虫。具有新毒素的病毒工程的最新进展 作为生物杀虫剂的基因改善性能引起了新的担忧 关于寄主范围和关于非目标生物的安全性， 包括人类。正确评估杆状病毒的安全性要求 对控制寄主的分子机制的进一步了解 范围和病毒复制。这项提议的长期目标是 检测最早的杆状病毒基因的功能和调控 定义主机与病毒相互作用和病毒复制的机制 战略。我们将主要关注早期的35K蛋白(P35)基因。 由原型杆状病毒--加州拟革兰氏菌编码 核型多角体病毒(AcMNPV)。P35对于 病毒以细胞特有的方式复制。它还可以防止早产。 宿主细胞裂解是病毒诱导的程序性细胞死亡的结果 (细胞凋亡)。细胞凋亡是一种内在的、信号诱导的过程，通过这种过程， 细胞自毁；它用于控制细胞数量。打压 细胞凋亡导致异常的细胞存活，因此有助于 致癌作用。其中涉及的机制尚不清楚。AcMNPV是第一个 已知可诱导非免疫性来源细胞凋亡的病毒和 首次同时编码抑制这种宿主的基因(P35) 回应。P35突变体的生成非常容易，而且 培养反应细胞的相对便利性提供了新的 有机会研究程序性细胞死亡的调节和 研究细胞凋亡作为抗病毒防御的可能作用 策略。在本提案中，我们使用了站点定向的组合 通过诱变和新型重组病毒研究其作用机制 其中p35阻断AcMNPV感染细胞的凋亡。遗传和 将使用生化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，以便继续定义顺应性 涉及的序列和反式作用因子(宿主和病毒) 杆状病毒转录调控。我们在这里关注的是监管 启动子上游活化区5‘端的基序 非编码RNA前导和病毒增强剂，每一种都已知会影响早期 抄写。最后，在证明了p35基因可以 作为一种强大的可选择标记(杆状病毒的第一个标记)，我们 开发一种新的基于转座子的系统，用于随机诱变 AcMNPV基因组对病毒基因的继续研究至关重要 组织和职能。总而言之，这些研究可望 对真核基因的复杂机制有了重要的见解 程序性细胞的调控以及诱导和抑制 与控制细胞增殖有关的死亡。