杆状病毒IE1在晚期基因表达中的作用研究

Early gene expression and late gene expression of baculoviruses rely on the host RNA polymerase II and a virus-encoded RNA polymerase respectively. The mechanism for initiation of late gene transcription has not been elucidated. IE1 is a multiple functional viral protein expressed in immediate early stage of infection. It is required for activation of viral early gene expression and DNA replication, and is one of the identified late expression factors. The role it plays in late virus late gene expression is not clear yet. In a previous study, we found that Autographa californica multiple nucleopolyhedrovirus IE1 is possibly involved in late gene expression directly. In this project, we plan to verify the role of IE1 in activation of virus late gene transcription by detection of the transcripts of some late virus genes in cell culture transfected with individual ie1 transient expression plasmids or by AcMNPV recombinants with the genes of the subunits of RNA polymerase knocked out, and identification of the transcription start site. Subsequently, we are going to identify the domains in IE1 associated with activation of late gene transcription. In further, experiments will be performed to identify interaction between IE1 and additional viral proteins or host insect proteins, and investigate effects of interaction between IE1 and addition proteins on late gene expression. The project intends to find out the role of AcMNPV IE1 in late gene expression. Completion of the project would contribute to reveal the mechanism of late gene expression initiation and regulation of baculoviruses and provide theoretical bases for improvement of the viruses by genetic engineering for application.

杆状病毒早期基因表达和晚期基因表达分别依赖宿主RNA聚合酶和其自身编码的RNA聚合酶。其晚期基因转录起始和调控机制尚未明了。IE1是一个极早期表达的多功能病毒蛋白，是病毒早期基因激活和DNA复制必需因子，也是已知的晚期表达因子之一。然而其在晚期基因表达中的作用尚未阐明。本项目拟在前期发现IE1可能直接参与晚期基因表达激活的基础上通过对苜蓿银纹夜蛾核多角体病毒(AcMNPV) RNA基因缺失突变体和ie1相关克隆质粒转染细胞培养的病毒晚期基因转录产物及其转录起始位点的测定验证IE1对病毒晚期基因的激活作用；识别IE1激活晚期基因表达的相关功能域或结构域；筛查和鉴定IE1的互作蛋白并分析IE1与其它蛋白的相互作用对晚期基因表达的影响。通过这些研究工作揭示IE1在杆状病毒晚期基因表达中的作用及作用机制。本项目研究工作的完成将推进对杆状病毒晚期基因表达机制的阐释，为杆状病毒的改良和利用提供理论依据

杆状病毒早期基因表达和晚期基因表达分别依赖于宿主RNA聚合酶II和病毒编码的RNA聚合酶；IE1是病毒早期基因表达、DNA复制和晚期基因表达必需因子；但其作用机制尚未明了。在本项目执行过程中，我们通过对苜蓿银纹夜蛾核多角体病毒（Autographa californica multiple nucleopolyhedrovirus, AcMNPV）RNA聚合酶亚基基因缺失突变体和ie1基因缺失突变体对Sf9细胞系的转染-感染和晚期基因表达实验分析，确定AcMNPV RNA聚合酶亚基基因缺失突变体不能完成子代病毒复制，但存在低水平晚期基因表达；而ie1基因缺失突变体既不能完成病毒复制，也没有晚期基因表达；通过构建AcMNPV 基因组分段克隆、亚克隆、单基因克隆和瞬时表达实验分析，发现AcMNPV ie1是唯一能够独立激活晚期基因表达的病毒因子基因；发现IE1激活晚期基因表达对宿主RNA聚合酶抑制剂不敏感；通过对AcMNPV RNA聚合酶亚基基因缺失突变体和基因组片段克隆的晚期基因转录产物分析，确定IE1激活晚期基因从非特异位点起始转录；在此基础上，通过对IE1编码序列的渐进截短和瞬时表达实验分析，完成了对其与晚期基因表达相关的功能域序列识别。基于这些实验结果，推测IE1激活晚期基因从非特异性位点起始转录可能为病毒RNA聚合酶募集于晚期基因启动子位点创造适宜的DNA构象。这些研究结果是对杆状病毒晚期基因表达和IE1的作用机制研究的新发现。此外，我们还确认AcMNPV 同源重复区（homologous repetitive region, hr）序列对IE1激活晚期基因表达也具有增强作用；通过免疫共沉淀联合质谱分析，鉴定出与IE1相互作用的7种其它病毒蛋白和7种宿主蛋白。本项目的研究结果深化了对杆状病毒晚期基因表达调控机制以及IE1在晚期基因表达中的作用的认识。