STRUCTURE AND FUNCTION OF POXVIRUS GENES

目的是确定痘病毒控制的机制， 他们的基因表达。 具体目标如下。 a）为了表征编码160K蛋白的牛痘病毒基因， 病毒诱导的A型包涵体的主要成分。 160K 蛋白质是晚期基因的产物，它是最丰富的蛋白质之一。 病毒蛋白合成。 基因的结构将由 通过核苷酸序列分析;以及转录的位点， 将被识别。 将使用定点诱变， 为了鉴定参与转录调节的顺式作用信号， 并研究该基因产物的功能。 B）表征编码非结构38 K的牛痘病毒基因， 蛋白 这种蛋白质在感染早期合成， 早期表达最强烈的基因之一。 的位点 将确定该基因的转录是否启动。 定点 然后将使用诱变来鉴定控制这些突变的区域。 转录的时间调节和控制转录速率的区域 转录。 还将检查蛋白质的功能。 的 38K蛋白可以指导病毒诱导的出血的产生， 病变 此外，该蛋白质的预测氨基酸序列共享 与丝氨酸抑制剂蛋白质的某些相似性 蛋白酶 因此，38K蛋白影响出血的能力 通过丝氨酸蛋白酶的抑制来检测。 （c）开发遗传互补系统，用于分析 痘病毒基因的功能。 将尝试获得细胞（辅助细胞）。 细胞），其将表达痘病毒的单个基因。 两种方法 将被用来实现这一点：一个将采用逆转录病毒表达 含有感兴趣的痘病毒基因的载体;第二种将使用 质粒，其中痘病毒基因在 基因调控的启动子元件。 表达a的辅助细胞 然后将特定的病毒基因用作分离的宿主细胞， 培养病毒，其中病毒基因组中的等同基因具有 被灭活了。 这些系统将用于以下方面：i） 试图分离出具有重要基因功能的病毒 ii）检查改变病毒的量的影响， - 相对于其他病毒蛋白的量，细胞中的病毒蛋白的量;以及 iii）检查在一定的温度下合成病毒基因产物的效果， 病毒生长周期中不正常产生的时期。

The objective is to identify the mechanisms by which poxviruses control their gene expression. The specific aims are as follows. a) To characterize the cowpox virus gene encoding the 160K protein that is the major component of the virus-induced A-type inclusions. The 160K protein is the product of a late gene, and it is one of the most abundant viral proteins synthesized. The structure of the gene will be determined by nucleotide sequence analysis; and the site at which transcrption is initiated will be identified. Site-directed mutagenesis will be used both to identify cis-acting signals involved in the regulation of transcription, and to investigate the functions of the gene product. b) To characterize the cowpox virus gene that encodes a non-structural 38K protein. This protein is synthesized early in infection, and is a product of one of the most strongly expressed early genes. The site at which trasnscription of this gene is initiated will be determined. Site-directed mutagenesis will then be used to identify the regions controlling the temporal regulation of transcription and regions controlling the rate of trasnscription. The function of the protein will also be examined. The 38K protein can direct the production of haemorrhage in virus-induced lesions. Moreover the predicted amino acid sequence of this protein shares some similarity to those of proteins that are inhibitors of serine proteases. Therefore the ability of the 38K protein to effect haemmorhage by the inhibition of serine proteases will be examined. c) To develop genetic complementation systems for use in analyses of the functions of poxvirus genes. Attempts will be made to obtain cells (helper cells) that will express individual genes of poxviruses. Two approaches will be used to achieve this: one will employ retrovirus expression vectors that contain the poxvirus gene of interest; the second will use plasmids in which the poxvirus gene is under the control of a hormonally-regulated promoter element. The helper cells expressing a particular virus gene will then be used as host cells for the isolation and culture of viruses in which the equivalent gene in the virus genome has been inactivated. These systems will be used in the following way: i) to try to isolate viruses that have had an essential gene function inactivated; ii) to examine the effects of altering the amounts of a viral protein in a cell, relative to the amounts of other viral proteins; and iii) to examine the effects of synthesizing a viral gene product at a period in the viral growth cycle when it is not normally produced.