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 transcrib ed during latency and is located antisense to the gamma(1)34.5 gene, E arlier studies have shown that the expression of ORF P is repressed by an infected cell protein no, I binding site straddling the transcript ion initiation site, We have made monospecific polyclonal antibodies t o the protein and constructed a virus, designated ORF P++, in which th e infected cell protein no, 4 binding site has been mutagenized, there by allowing full expression of an unmodified ORF P gene from its natur al promoter, We report the following findings, (i) The native protein forms multiple bands on denaturing polyacrylamide gels suggestive of e xtensive processing and aggregation of the protein; (ii) the protein a ccumulates in the nucleus in rod-shaped structures perpendicular to th e axis of attachment of the infected cell to the solid matrix; (iii) t he virus was highly attenuated on inoculation into mice by the intrace rebral or ocular route, and virus was not recovered upon explantation of trigeminal ganglia; (iv) although protein synthesis was not prematu rely shut off in the human neuroblastoma cell line SK-N-SH, gamma(1)31 .5 protein was not detected in immunoblots. Analyses of electrophoreti cally separated denatured RNAs indicated that in cells infected with t he ORF P++ virus, there was a large increase in the amount of ORF P RN A and a corresponding decrease in the amount of gamma(1)31.5 RNA. We c onclude that either the overproduction of ORF P protein blocks the exp ression of some herpes simplex virus 1 genes or derepression of the tr anscription of ORF P has a negative effect on the transcription of the antisense, gamma(1)34.5 RNA.