Phenotypic properties of herpes simplex virus 1 containing a derepressed open reading frame P gene

Open reading frame P (ORF P) maps in the viral DNA sequences transcribed during latency and is located antisense to the gamma(1)34.5 gene, Earlier studies have shown that the expression of ORF P is repressed by an infected cell protein no, I binding site straddling the transcription initiation site, We have made monospecific polyclonal antibodies to the protein and constructed a virus, designated ORF P++, in which the infected cell protein no, 4 binding site has been mutagenized, thereby allowing full expression of an unmodified ORF P gene from its natural promoter, We report the following findings, (i) The native protein forms multiple bands on denaturing polyacrylamide gels suggestive of extensive processing and aggregation of the protein; (ii) the protein accumulates in the nucleus in rod-shaped structures perpendicular to the axis of attachment of the infected cell to the solid matrix; (iii) the virus was highly attenuated on inoculation into mice by the intracerebral or ocular route, and virus was not recovered upon explantation of trigeminal ganglia; (iv) although protein synthesis was not prematurely shut off in the human neuroblastoma cell line SK-N-SH, gamma(1)31.5 protein was not detected in immunoblots. Analyses of electrophoretically separated denatured RNAs indicated that in cells infected with the ORF P++ virus, there was a large increase in the amount of ORF P RNA and a corresponding decrease in the amount of gamma(1)31.5 RNA. We conclude that either the overproduction of ORF P protein blocks the expression of some herpes simplex virus 1 genes or derepression of the transcription of ORF P has a negative effect on the transcription of the antisense, gamma(1)34.5 RNA.