REPRESSION OF THE HERPES-SIMPLEX VIRUS-1 ALPHA-4 GENE BY ITS GENE-PRODUCT OCCURS WITHIN THE CONTEXT OF THE VIRAL GENOME AND IS ASSOCIATED WITH ALL 3 IDENTIFIED COGNATE SITES

The infected cell protein 4 (ICP-4), the major regulatory protein encoded by the a4 gene of the herpes simplex virus 1, binds two sites (alpha4-1 proximal, alpha4-1 distal) at the 5'-untranscribed domain and at the transcription initiation site (alpha4-2) of the alpha4 gene. Chimeric genes consisting of the 5'-untranscribed and transcribed noncoding domains of the alpha4 gene fused to the coding sequences of the thymidine kinase gene were mutagenized to abolish binding of ICP-4 by substitution of bases, including the guanines whose methylation interferes with binding of the protein, and recombined into the viral genome. The cytoplasmic RNAs extracted from infected cells treated with cycloheximide, from untreated infected cells maintained for 4 or 8 hr, and from cells infected first with a virus deleted in the alpha22 gene and 3 hr later with the test viruses were tested in RNase protection assays for amounts of the chimeric gene RNA relative to amounts of alpha22 gene RNA. We report the following: (i) Mutation of the alpha4-2 binding site resulted in a 5-to 6-fold higher accumulation of chimeric gene RNA at 4 hr and as much as 15-fold higher accumulation by 8 hr after infection. (ii) Mutations of alpha4-1 sites by themselves had no effect on RNA accumulation. However, mutagenesis of all three sites significantly increased mRNA amounts above the levels seen in cells infected with alpha4-2 site mutants. (iii) The mutations have no effect on accumulation of alpha4 mRNA in the absence of ICP-4 synthesis and, therefore, the mutations had no effect on RNA stability or transcription rate. (iv) Accumulation of alpha4 mRNA relative to that of alpha22 mRNA is highest in the presence of cycloheximide and decreases with time after infection. We conclude that ICP-4 autoregulates the transcription of its own gene in infected cells and that binding of ICP-4 to three sites in its promoter is additive in its effects on this process.