THE ROLE OF ICP4 REPRESSOR ACTIVITY IN TEMPORAL EXPRESSION OF THE IE-3 AND LATENCY-ASSOCIATED TRANSCRIPT PROMOTERS DURING HSV-1 INFECTION

The herpes simplex virus (HSV) type 1 immediate-early protein ICP4 rep resses transcription of its own gene and possibly that of other immedi ate-early genes. In the present study, we analyzed the role that an IC P4 binding site present in two HSV-1 promoters plays in the level and kinetics of expression during HSV-1 infection. Wild-type and mutant fo rms of the IE-3 (ICP4) promoter and the latency associated promoter (L AP) were fused to the thymidine kinase (tk) coding sequences and trans ferred to the genome of an 1CP4-deficient virus. Promoter mutants were constructed to assess the effect of the ICP4 binding site in the pres ence and absence of defined binding sites for cellular and other viral factors in the promoters. The activities of the promoter constructs w ere inferred from the level of tk mRNA seen following viral infection in the absence of ICP4, in Vero cells and in the presence of ICP4 in I CP4 expressing E5 cells. Kinetics of expression and the dependence on DNA synthesis for expression were examined following infection of 55 c ells. In the presence of the ICP4 binding site in LAP and in IE3 promo ters lacking TAATGARAT motifs, expression was maximal late after infec tion and was greatly reduced when DNA synthesis was inhibited. When th e ICP4 binding site was removed, both LAP and the IE3 construct retain ing an Sp1 site were more abundantly expressed and exhibited kinetics of expression indistinguishable from that of the tk promoter. in vitro , ICP4 repressed LAP transcription mediated by the general transcripti on factors and upstream activating proteins. Deletion of the ICP4 bind ing site not only relieved repression, but in the presence of USF acti vity, ICP4 further induced LAP transcription. The results of these exp eriments suggest a role for the repressor activity of ICP4 in the temp oral regulation of HSV-1 transcription. (C) 1994 Academic Press, Inc.