TRANSCRIPTION OF THE DEREPRESSED OPEN READING FRAME-P OF HERPES-SIMPLEX-VIRUS-1 PRECLUDES THE EXPRESSION OF THE ANTISENSE GAMMA(1)34.5 GENEAND 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 antisen se to the gamma(1)34.5 gene, is naturally repressed by infected cell p rotein 4 (ICP4), the major herpes simplex virus 1 regulatory protein. Earlier studies on cells infected with a mutant in which the expressio n 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 protei n colocalizes with spliceosomes, (iii) both gamma(1)34.5 mRNA and prot ein are virtually undetectable, and (iv) the virus is attenuated on in tracerebral inoculation in mice. We report the construction and charac terization of two recombinant viruses: R7546, in which ORF P transcrip tion was derepressed and the initiator methionine codon was replaced; and R7547, in which both mutations were repaired to the wild-type geno type. The mutations in R7546 do not alter the amino acid sequence of t he gamma(1)34.5 gene. We report that (i) the reduction in the accumula tion of gamma(1)34.5 mRNA and protein in cells infected with mutant vi ruses expressing derepressed ORF P genes reflects the effects of antis ense 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 exp ression of ORF P protein.