HERPESVIRUS GENE EXPRESSION IN TRANSFORMED CELLS

本项目的长期目标是确定 单纯疱疹病毒转化细胞中病毒基因的表达 （HSV）。 注意力将首先集中在HSV基因的调控上， 胸苷激酶和一种膜糖蛋白，因为 转化的细胞系衍生物的表达水平不同， 这些基因。 这项建议的目的之一是确定是否改变 在转化细胞中病毒基因的表达与缺失相关 或病毒DNA序列的重排。 第二个目标是描述 从病毒DNA的选定区域产生的mRNA的结构特征 序列，与生产性感染过程中产生的mRNA相比， 为了确定反映不同的结构差异， 转录和转录后过程所需的生产一个 转化和感染细胞中的功能性病毒基因产物。 第三个目标是 为了进一步研究提高常驻病毒基因水平的基础， 表达在转化细胞超感染HSV，也为最近 发现转化细胞中某些细胞蛋白质的合成， HSV超感染增强，与细胞增殖抑制相反， 通常伴随HSV感染的蛋白质合成。 所用方法 包括用限制性内切核酸酶消化转化细胞DNA， 通过电泳分离并转移至硝酸纤维素过滤器， 通过含有病毒DNA序列的片段的杂交鉴定。 病毒mRNA将通过与病毒DNA片段杂交而富集， 纤维素，通过估计大小通过电泳分级，和 恢复的单个物种，以精确识别病毒DNA序列， 它们都是同源的，并在体外测试信使活性。 的 特异性病毒和细胞蛋白的表达 转化的细胞将通过酶测定和免疫沉淀来监测 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.