Temporal Regulation of Baculovirus Gene Expression

苜蓿夜蛾核型多角体病毒的多角体蛋白基因在细胞外病毒释放后的很晚感染阶段表达最强。人们认为，多角体蛋白的时间调控需要一个即刻早期基因(S)的顺序表达来激活延迟的早期基因表达；一个延迟的早期基因(S)来开启DNA合成和大基因表达；以及一个晚基因(S)来刺激多角体蛋白，这是一个非常大的基因。我设计了一种互补分析方法来定位控制多角体蛋白表达的时间调控的病毒因素。构建了多角体蛋白启动子控制下的细菌基因氯霉素乙酰转移酶(CAT)的表达载体polcat。CAT在允许的昆虫细胞与Polcat和病毒DNA共转染时表达，但不与先前用限制性内切酶消化的病毒DNA共转染。消化后的DNA不能支持polcat的表达，显然是由于限制性内切酶使一个或多个必需基因失活。这些基因的位置可以通过与克隆的PstI-限制性片段互补来定位。初步结果表明，12个PstI克隆补充了病毒DNA的不同消化，表明至少有12个基因参与了时间级联。我打算绘制并确定这些因素中的几个在多角体蛋白表达的时间调控中的功能。这个项目的最终目标是定义一组有限的病毒基因，允许在昆虫细胞中高水平表达多角体蛋白。虽然在授权期间可能不可能确定所有的因素，但这项研究应该会增加我们对杆状病毒基因表达的时间调节机制的理解。

The polyhedrin gene of Autographa californica nuclear polyhedrosis virus is maximally expressed very late in infection, after the release of extracellular virus. It is believed that temporal regulation of polyhedrin requires the sequential expression of an immediate early gene(s) to activate delayed early gene expression; a delayed early gene(s) to turn on DNA synthesis and large gene expression; and a late gene(s) to stimulate polyhedrin, a very large gene. I have devised a complementation assay to map the viral factors which control the temporal regulation of polyhedrin expression. A plasmid, polcat, was constructed that contains the bacterial gene chloramphenicol acetyltransferase (CAT) under the control of the polyhedrin promoter. CAT is expressed upon cotransfection of permissive insect cells with polcat and viral DNA, but not with viral DNA which has previously been digested with restriction enzymes. The inability of digested DNA to support polcat expression is apparently due to inactivation of one or more essential genes by restriction digest. The location of these genes can be mapped by complemenation with cloned PstI-restriction fragments. Preliminary results indicated that twelve of the PstI clones complement different digests of viral DNA, indicating that at least twelve genes are involved in the temporal cascade. I intend to map and determine the function of several of these factors in temporal regulation of polyhedrin expression. The ultimate goal of this project is to define a limited set of viral genes which permits high level expression of polyhedrin in insect cells. Although it may not be possible to identify all of the factors during the grant period, this research should increase our understanding of the mechanisms of temporal regulation of baculovirus gene expression.