The analysis of the regulatory phenomena controlling the coordinately and sequentially ordered synthesis of herpes simplex virus type I (HSV-1) specific proteins during infection continues to be the objective of this research. Two new initiatives are proposed. The first is an extension of previous studies on the regulation of expression of the glycoprotein C (gC) gene, a late gene. Late gene expression requires transactivation by the gene products of two immediate-early genes, ICP4 and ICP27, and has a strict requirement for the synthesis of viral DNA for expression. The cis-acting regulatory element controlling gC transcription is a 15 bp sequence centered around the TATA box. The requirement for DNA synthesis can be alleviated by fusing the upstream transcriptional regulatory sequences of the early tk gene 5' to the gC TATA element. The requirement for viral DNA synthesis for late gene expression will be the main focus of these experiments. The minimum transcriptional signal capable of giving DNA replication independent expression of the gC gene will be determined. The hypothesis that ICP8, the HSV-1 major DNA binding protein, functions as a general repressor of late gene expression prior to DNA replication will be tested. Using a virus carrying the tk-gC chimeric gene and a temperature sensitive mutant allele of the alpha 27 gene, the role of ICP27 in late gene expression will be probed. Studies have been initiated on the regulation of expression of the gene for the HSV-1 specified large subunit of ribonucleotide reductase (RR1). The RR1 gene shows characteristics of both immediate-early and early gene regulation and, in contrast to all other HSV-1 genes, may be uniquely transactivated by ICP0 instead of ICP4 during infection. The hypothesis will be tested that in infection immediate-early expression is achieved through alphaTIF and the octamer/TAATGARATTC element, while early expression is accomplished through ICP0 and the downstream regulatory signals of the RR1 promoter. The cis-acting signals critical for transactivation by alpha TIF and ICP0 will be defined. Exchange of cis-acting signals between the RR1 promoter and the ICP4 responsive tk promoter should identify the sequences responsive to induction by ICP0 and ICP4. Experiments are proposed to determine if the alpha0 gene product is the only viral transactivator regulating RR1 expression during the early phase of infection. Finally, the RR1 promoter mutant viruses will be tested in mice for effects on neurovirulence and on the establishment and reactivation from latency.
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