Temporal Regulation of Baculovirus Gene Expression

病毒使用许多不同的策略来接管宿主细胞，迫使它们合成病毒蛋白质和核酸，而不是细胞生存所需的宿主分子。杆状病毒做到了这一点，部分是通过合成自己的RNA聚合酶来实现的，这种聚合酶是为新病毒颗粒制造蛋白质所必需的。这是一个简单的四亚基复合体，但它具有识别病毒启动子、转录相关基因、终止转录以及处理转录RNA的5‘和3’端的能力。该项目的总体目标是了解病毒转录的分子机制，重点是调节病毒RNA聚合酶活性的因素。该项目的智力价值将通过完成四个具体目标来实现。第一个是确定转录因子Lef-5的功能，它与细胞延伸因子相似，但似乎在启动过程中起作用。这个项目将通过分析流产的起始产物和向延长的过渡来检验Lef-5刺激启动子逃逸的假设。第二个目标是完成病毒mRNA封闭酶的鉴定，并描述MTase-1的附加功能。杆状病毒RNA聚合酶和MTase1蛋白占形成mRNA5‘帽所需的四种活性中的三种。催化剩余步骤的宿主或病毒蛋白将通过对该活性的纯化来鉴定，该活性是在病毒感染时诱导的。第三个目的是通过分离转录起始的不同步骤的生化分析来了解启动子特异性因子VLF-1的分子机制。最后，我们将探讨Lef-6和Lef-10在晚期基因表达调控中的作用。为此，将对这些蛋白进行表达和纯化，并研究它们与病毒RNA聚合酶的相互作用。此外，将通过对突变病毒的分析来研究这些蛋白质在体内的作用。除了这个项目的发现方面，这里描述的工作将为德克萨斯农工大学生物技术硕士项目的学生提供一个极好的培训机会。这是一个新的专业科学硕士项目，强调学生在生物技术方面的体验式培训，课程包括商业和新闻。随着生物科学行业难以用高技能人才填补理科硕士级别的职位，这类项目的重要性预计会越来越大。来自国家科学基金会的支持将提供基本技术培训的机会，这将提高学生在分子生物技术方面的能力，并增强他们找到具有挑战性和回报的工作的能力。因此，该项目更广泛的价值在于促进发现和理解，同时促进传统和非传统研究生课程的教学、培训和学习。

Viruses use a number of different strategies to take over host cells and force them to synthesize viral proteins and nucleic acids instead of the host molecules that are needed for cell survival. Baculoviruses do this, in part, by synthesizing their own RNA polymerase that is needed to make proteins for new virus particles. This is a simple four-subunit complex, yet it has the ability to recognize viral promoters, transcribe linked genes, terminate transcription, and process 5' and 3' ends of transcribed RNAs. The overall objective of this project is to understand the molecular mechanisms of viral transcription, focusing on the factors that regulate the activity of viral RNA polymerase. The intellectual merit of the project will be achieved through the completion of four specific objectives. The first is to define the function of the transcription factor LEF-5, which has similarity to cellular elongation factors, yet appears to act during initiation. This project will test the hypothesis that LEF-5 stimulates promoter escape, through the analyses of abortive initiation products and the transition to elongation. The second objective is to complete the identification of viral mRNA capping enzymes and describe additional functions of MTase-1. Baculovirus RNA polymerase and the protein MTase1 account for three of four activities required for the formation of mRNA 5' caps. The host or viral protein that catalyzes the remaining step will be identified through purification of this activity, which is induced upon viral infection. The third objective is to understand the molecular mechanism of the promoter specificity factor VLF-1 through biochemical analyses that separate the different steps of transcription initiation. Finally the roles of LEF-6 and LEF-10 in the regulation of late gene expression will be investigated. To this end, the proteins will be expressed and purified, and their interactions with viral RNA polymerase investigated. Further, the in vivo roles of these proteins will be studied through analyses of mutant viruses. In addition to the discovery aspects of this project, the work described here will provide an excellent training opportunity for students in the Master of Biotechnology program at Texas A&M University. This is a new Professional Science Master's program that emphasizes student experiential training in biotechnology, with coursework in business and journalism. These types of programs are expected to grow in importance, as the bioscience industry struggles to fill positions at the M.S. level with highly skilled personnel. Support from the NSF will provide the opportunity for training in basic techniques that will increase students' competency in molecular biotechnology and enhance their ability to find jobs that are challenging and rewarding. Thus, the broader merit of the project lies in the advancement of discovery and understanding while promoting teaching, training, and learning in traditional and non-traditional graduate programs.