HERPESVIRUS GENE EXPRESSION IN TRANSFORMED CELLS

该项目的长期目标是定义决定因素 单纯疱疹病毒转化细胞中病毒基因的表达 (HSV)。最初的注意力将集中在HSV基因的调节上 胸苷激酶和一种膜糖蛋白，因为可获得 表达水平不同的转化细胞系衍生品 这些基因。这项提案的目的之一是确定变化是否 病毒基因在转化细胞中的表达与缺失相关 或病毒DNA序列的重排。第二个目标是刻画 病毒DNA部分区域产生的mRNAs的结构特征 序列，与在生产性感染过程中产生的mRNA相比，在 以确定反映 转录和转录后过程是产生一种 转化和感染细胞中的功能性病毒基因产物。第三个目标是 进一步探讨常驻病毒基因水平升高的基础 单纯疱疹病毒重叠感染转化细胞中的表达 发现转化细胞中某些细胞蛋白质的合成 与单纯疱疹病毒重叠感染对细胞的抑制相反 通常伴随单纯疱疹病毒感染的蛋白质合成。应使用的方法 包括用限制性内切酶消化转化细胞DNA， 通过电泳法进行分级，并转移到硝酸纤维素滤器中 通过杂交含有病毒DNA序列的片段进行鉴定。 通过与病毒DNA片段杂交，病毒mRNAs将得到丰富 纤维素，通过电泳法通过大小估计进行分级，以及 恢复单个物种以准确识别病毒DNA序列 每一个都是同源的，并在体外测试信使活性。这个 鸡传染性支气管炎病毒重叠感染后特异性病毒和细胞蛋白的表达 转化的细胞将通过酶分析和免疫沉淀进行监测。 用同基因仓鼠产生的抗HSV转化仓鼠的抗血清 细胞。

The long-range objective of this project is to define the factors that govern the expression of viral genes in cells transformed by herpes simplex virus (HSV). Attention will be focused initially on regulation of the HSV genes for thymidine kinase and a membrane glycoprotein, because of the availability of transformed cell line derivatives that differ in the levels of expression of these genes. One of the aims of this proposal is to determine whether changes in the expression of a viral gene in transformed cells correlate with deletions or rearrangements of the viral DNA sequences. A second aim is to characterize structural features of mRNAs produced from selected regions of the viral DNA sequences, in comparison with the mRNAs made during productive infection, in order to identify structural differences that reflect differences in the transcriptional and post-transcriptional processes required for production of a functional viral gene product in transformed and infected cells. A third aim is to investigate further the basis for enhanced levels of resident viral gene expression in transformed cells super-infected with HSV and also for a recent finding that synthesis of some cellular proteins in transformed cells is enhanced by HSV super-infection, in contrast to the inhibition of cellular protein synthesis that usually accompanies HSV infection. Methods to be used include digestion of transformed cell DNA with restriction endonucleases, fractionation by electrophoresis and transfer to nitrocellulose filters for identification by hybridization of fragments containing viral DNA sequences. Viral mRNAs will be enriched by hybridization to viral DNA fragments on cellulose, fractionated by electrophoresis by estimations of size, and individual species recovered to identify precisely the viral DNA sequences to which each is homologous and to test messenger activity in vitro. The expression of specific viral and cellular proteins after super-infection of transformed cells will be monitored by enzyme assays and by immunoprecipitation with antisera produced in syngeneic hamsters against HSV-transformed hamster cells.