The aims of this research project are to understand the regulation of viral gene expression during herpesvirus infection, evaluate the feasibility of herpesvirus vectors as gene therapy vectors, and determine the herpesvirus genes that would be most effective for development as potential subunit vaccines. Understanding the regulation of HSV gene expression is essential for the development and evaluation of successful vaccines, particularly live virus vaccines. During herpes simplex virus infection, approximately 75 genes are expressed in a well ordered process that can be conveniently divided into three phases based on the temporal order of mRNA synthesis. Immediate-early genes are expressed soon after virus infection, and their gene products are required for the expression of early genes, many of whose gene products are involved in viral DNA replication. Expression of viral late genes requires viral DNA replication and functional immediate-early gene products. In FY99, the first phase of a genetic approach to identify factors that regulate HSV temporal gene expression was completed. In this approach, a temperature-sensitive cell line, ts13, that is defective for the eukaryotic transcription factor TAF250 at the non-permissive temperature was used to determine the role of this factor in HSV transcription. Results showed that immediate-early genes were transcribed at normal levels at the non-permissive temperature, the transcription of early genes was slightly reduced, and transcription of late genes was almost completely abolished. Interestingly, DNA replication did occur in these cells at the non-permissive temperature. Thus, the failure of HSV-1 to replicate in ts13 cells at the non-permissive temperature is probably due to a defect in late gene expression. A second part of this research project is designed to evaluate the feasibility of replication-incompetent herpes simplex viruses as gene therapy vectors. Studies during FY99 have focused on construction of recombinant vectors that can be induced to express foreign genes in the presence of the antibiotic tetracycline. Several novel vectors were constructed containing tet-inducible elements linked to HSV-1 immediate-early promoter elements. These vectors will be evaluated for their ability to express foreign genes in the presence of tetracycline. The third part of this research project is designed to evaluate various HSV gene products as potential components of subunit vaccines. At the present time, experiments are underway to clone clone multiple HSV-2 genes and to evaluate their ability to elicit a protective immune response when delivered simultaneously by DNA immunization.
