Temporal Regulation of Baculovirus Gene Expression

杆状病毒在真核DNA病毒中是独特的，因为早期基因由宿主RNA聚合酶II转录，而晚期和极晚期基因由病毒编码的RNA聚合酶转录。病毒RNA聚合酶是一种具有内在启动子识别、mRNA加帽、终止和多聚腺苷酸化活性的四亚基复合物。该项目的第一个目标是鉴定参与病毒RNA上5'帽形成的帽甲基转移酶。将使用两种互补的方法;一种是从感染的细胞中纯化cap甲基转移酶;另一种是测试orf 69蛋白的cap甲基转移酶活性。该蛋白是rRNA甲基转移酶FtzJ的同系物，是唯一具有S-腺苷甲硫氨酸结合基序的病毒蛋白。该项目的第二个目的是定义允许RNA聚合酶区分晚期和非常晚期模板的机制。同样，将使用两种方法来解决这个问题。首先，将对VLF-1的体外活性进行靶向分析，VLF-1在体内刺激非常晚的基因表达。然后，改变RNA聚合酶启动子特异性的因子将从感染的细胞中纯化。该项目的第三个目的是表征杆状病毒蛋白LEF-5，其与转录延伸因子TFIIS具有同源性。为了验证LEF-5是否具有延伸因子的功能，将进行实验，以确定LEF-5是否与RNApol结合，以及是否刺激RNApol的RNA切割活性;这两种活性是TFIIS的特征，也是其功能所必需的。该项目将阐明调节杆状病毒基因时间表达的机制，也应该对生物技术产生实际影响。杆状病毒表达系统已用于生产数千种用于基础研究和商业项目的不同蛋白质。 杆状病毒RNA聚合酶是该表达系统的核心，对其独特性质的理解将导致该技术的改进。

Baculoviruses are unique among eukaryotic DNA viruses because the early genes are transcribed by the host RNA polymerase II, while late and very late genes are transcribed by a viral-encoded RNA polymerase. The viral RNA polymerase is a four subunit complex with intrinsic promoter recognition, mRNA capping, termination, and polyadenylation activities. The first goal of this project is to identity the cap methyltransferase that participates in the formation of 5' caps on viral RNAs. Two complementary approaches will be used; one is to purify cap methyltransferase from infected cells; the other is to test orf69 protein for cap methyltransferase activity. This protein, which is a homolog of the rRNA methyltransferase FtzJ, is the only viral protein with an S-adenosyl-methionine binding motif. The second aim of this project is to define the mechanisms that allow RNA polymerase to discriminate between late and very late templates. Again, two approaches to this problem will be used. First, a targeted analysis of the in vitro activity of VLF-1, which stimulates very late gene expression in vivo, will be conducted. Then, factors that alter the promoter specificity of RNA polymerase will be purified from infected cells. The third aim of this project is to characterize the baculovirus protein LEF-5, which has homology with transcription elongation factor TFIIS. To test whether LEF-5 functions as an elongation factor, experiments will be performed to determine whether LEF-5 binds RNApol and whether it stimulates the RNA cleavage activity of RNApol; these two activities are characteristic of TFIIS and are required for its function.This project will elucidate the mechanisms that regulate temporal expression of baculovirus genes, and should also have practical impacts on biotechnology. The baculovirus expression system has been used to produce thousands of different proteins used for basic research and commercial projects. The baculovirus RNA polymerase is central to this expression system, and an understanding of its unique properties will lead to improvements to this technology.