Transcription of the derepressed open reading frame P of herpes simplex virus 1 precludes the expression of the antisense gamma(1)34.5 gene and may account for the attenuation of the mutant virus

Open reading frame P (ORF P), located at the 3' terminus of the 8.5-kb DNA sequence transcribed during latency and almost completely antisense to the gamma(1)34.5 gene, is naturally repressed by infected cell protein 4 (ICP4), the major herpes simplex virus 1 regulatory protein. Earlier studies on cells infected with a mutant in which the expression of ORF P is derepressed have shown that (i) the accumulation of the alpha infected cell proteins 0 (ICP0) and 22 (ICP22), the products of spliced mRNAs, is reduced congruent with the binding of ORF P protein to p32, a component of the ASF/SF2 splicing factors, (ii) ORF P protein colocalizes with spliceosomes, (iii) both gamma(1)34.5 mRNA and protein are virtually undetectable, and (iv) the virus is attenuated on intracerebral inoculation in mice. We report the construction and characterization of two recombinant viruses: R7546, in which ORF P transcription was derepressed and the initiator methionine codon was replaced; and R7547, in which both mutations were repaired to the wild-type genotype. The mutations in R7546 do not alter the amino acid sequence of the gamma(1)34.5 gene. We report that (i) the reduction in the accumulation of gamma(1)34.5 mRNA and protein in cells infected with mutant viruses expressing derepressed ORF P genes reflects the effects of antisense transcription of ORF P rather than a function of ORF P protein, (ii) the attenuated phenotype of the viruses carrying derepressed ORF P genes is due largely to the absence of the gamma(1)34.5 protein, and (iii) the reduction in accumulation of ICP0 and ICP22 requires the expression of ORF P protein.