Temporal Regulation of Baculovirus Gene Expression

杆状病毒基因表达的时间调控

• 批准号: 9506233
• 负责人: Linda Guarino
• 金额: $ 28万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506233&HistoricalAwards=false
• 关键词: Temporal; Regulation; Baculovirus; Gene; Expression

9506233 Guarino Baculovirus late and very late genes are expressed at different times post infection. The late genes encode viral structural proteins and their expression is maximal between 24 - 36 hr post infection. Polyhedrin is an example of the very late genes which are involved in the formation of occlusion bodies in the nuclei of infected cells. Transcription of the very late genes is relatively low at 24 hr and reaches maximal levels at 48 hr, after the synthesis of late proteins and assembly of virions. This suggests that the late and very late promoters are controlled by different factors, although the mechanisms that regulate differential transcription of the two classes of genes are unknown. The goals of this project are to purify and identify the viral proteins that are directly involved in transcription of the late and very late genes. For this research, hybrid templates have been constructed that contain late or very late promoters linked to a synthetic DNA fragment that lacks cytidine residues on the transcript strand. In vitro transcription reactions performed in the absence of CIP yield strong signals resulting from accurate initiation at the correct sites. These constructs were used to monitor the purification of active transcription complexes from infected cell extracts prepared at 36 hr post infection. Phosphocellulose chromatography yielded two fractions that differentially transcribed the late and very late templates. Addition of the follow-through fraction to either of the active fractions resulted in an increase in transcription from both late and very late promoters, although the flow-through fraction had no transcription activity when tested separately. The very late transcription complex was purified on three additional columns. The purified complex contains only five proteins and transcribed the polyhedrin and 39k C-free constructs in the absence of additional factors, indicating that the complex has both promoter recognition and RNA polymerase activities. Next, the late complex will be purified in order to compare the protein compositions of the two complexes. Proteins in the two complexes will be identified using a combination of methods including V8 protease mapping, immunochemical analyses, and direct protein sequencing. The DNA binding activities of the two complexes will be compared and proteins that differ between the two complexes will be targeted for further study, including an analysis of their promoter recognition. The factors that increase expression of late and very late genes will be purified and the corresponding genes identified. %%% This research should aid in understanding the mechanisms of transcriptional regulation of baculovirus late and very late gene expression. The baculoviruses late transcription system is unique among eukaryotic viruses. In some respects, it seems very primitive. The fact that late and very late promoters are preferentially transcribed by different RNA polymerase complexes is reminiscent of bacterial RNA polymerase and sigma factors. However, transcription is increased by the addition of separable factors, a strategy of eukaryotic transcription systems. In addition, results from this research may have a practical impacts on biotechnology. The baculovirus expression system is widely used for the production of large amounts of medically important proteins for use as vaccines, diagnostic tools, or therapeutic agents. However, the baculovirus system has limitations: it is transient because the viral vectors kill the cells; and recombinant proteins are expressed late when cellular processing pathways are compromised. A knowledge of the mechanisms that control baculovirus polyhedrin expression may aid in the design of vectors that work in the absence of virus infection and thus do not kill the host or impair protein processing. ***

瓜里诺杆状病毒晚期和极晚期基因在感染后的不同时间表达。晚期基因编码病毒结构蛋白，它们的表达在感染后24 - 36小时之间最大。多角体蛋白是参与在受感染细胞的细胞核中形成闭塞体的非常晚期基因的一个例子。在晚期蛋白质合成和病毒体组装后，极晚期基因的转录在24小时相对较低，并在48小时达到最高水平。这表明晚期和极晚期启动子受不同因素控制，尽管调节这两类基因差异转录的机制尚不清楚。该项目的目标是纯化和鉴定直接参与晚期和极晚期基因转录的病毒蛋白。为了这项研究，已经构建了杂交模板，其含有与转录物链上缺乏胞苷残基的合成DNA片段连接的晚期或极晚期启动子。在不存在CIP的情况下进行的体外转录反应产生强烈的信号，这是由于在正确的位点准确起始。这些构建体用于监测从感染后36小时制备的感染细胞提取物中纯化活性转录复合物。磷酸纤维素层析产生了两个馏分，差异转录的晚期和非常晚的模板。添加后续通过馏分的活性馏分中的任一个导致在转录从晚期和非常晚的启动子的增加，虽然流通馏分没有转录活性时，分别测试。在另外三个柱上纯化极晚期转录复合物。纯化的复合物仅含有5种蛋白质，并在不存在其他因子的情况下转录多角体蛋白和39 k无C构建体，表明该复合物具有启动子识别和RNA聚合酶活性。接下来，将纯化晚期复合物以比较两种复合物的蛋白质组成。将使用包括V8蛋白酶图谱、免疫化学分析和直接蛋白质测序的方法的组合来鉴定两种复合物中的蛋白质。将比较两种复合物的DNA结合活性，并将针对两种复合物之间不同的蛋白质进行进一步研究，包括分析其启动子识别。 将纯化增加晚期和极晚期基因表达的因子并鉴定相应的基因。 这项研究有助于理解杆状病毒晚期和极晚期基因表达的转录调控机制。杆状病毒的晚期转录系统是真核病毒中唯一的。在某些方面，它似乎非常原始。晚期和极晚期启动子优先被不同的RNA聚合酶复合物转录的事实让人想起细菌RNA聚合酶和σ因子。 然而，转录增加了可分离的因素，真核转录系统的策略。 此外，这项研究的结果可能对生物技术产生实际影响。 杆状病毒表达系统广泛用于生产大量医学上重要的蛋白质，用作疫苗、诊断工具或治疗剂。然而，杆状病毒系统具有局限性：它是瞬时的，因为病毒载体杀死细胞;当细胞加工途径受损时，重组蛋白表达较晚。控制杆状病毒多角体蛋白表达的机制的知识可能有助于设计在没有病毒感染的情况下工作的载体，从而不会杀死宿主或损害蛋白质加工。 ***