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 i s 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 i s 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.
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