REPRESSION OF THE HERPES-SIMPLEX VIRUS-1 ALPHA-4 GENE BY ITS GENE-PRODUCT (ICP4) WITHIN THE CONTEXT OF THE VIRAL GENOME IS CONDITIONED BY THE DISTANCE AND STEREOAXIAL ALIGNMENT OF THE ICP4 DNA-BINDING SITE RELATIVE TO THE TATA BOX

Infected cell protein no. 4 (ICP4), the major regulatory protein encod ed by the alpha 4 gene of herpes simplex virus 1, binds to a site (alp ha 4-2) at the transcription initiation site of the alpha 4 gene. An e arlier report described the construction of recombinant viruses that c ontained chimeric genes (alpha 4-tk) that consisted of the 5' untransc ribed and transcribed noncoding domains of the alpha 4 gene fused to t he coding sequences of the thymidine kinase gene and showed that disru ption of the alpha 4-2 binding site by mutagenesis derepressed transcr iption of this gene (N. Michael and B. Roizman, Proc. Natl. Acad. Sci. USA 90:2286-2290, 1993). This experimental design was used to determi ne the effect of displacement of the alpha 4-2 binding site on the rep ression of alpha 4 gene transcription by ICP4. We report the following findings. (i) In the absence of the alpha 4-2 binding site, at 4 h af ter infection, alpha 4-tk RNA levels increased 10-fold relative to the corresponding RNA levels of a gene that contained the alpha 4-2 site at its natural location. Displacement of the alpha 4-2 binding site by approximately one, two, and three turns of the DNA helix, i.e., by 10 , 21, and 30 nucleotides downstream of the original site, increased th e concentration of alpha 4-tk RNA 2.4-, 3.5-, and 5.8-fold, respective ly. (ii) Displacement of 16 nucleotides, i.e., approximately 1.5 helic al turns, increased the accumulation of alpha 4-tk by 5.3-fold, i.e., more than predicted by displacement alone. (iii) At 8 h after infectio n in the absence of the binding site, the accumulation of alpha 4-tk R NA increased 13.6-fold. However, in cells infected with recombinants t hat carried displaced alpha 4-2 binding sites, RNA accumulation decrea sed relative to the levels seen at 4 h after infection. The insertion of DNA sequences in order to displace the alpha 4-2 binding site had n o effect on accumulation of RNA in the presence of cyclohex-imide, i.e ., in the absence of ICP4, or on maximum accumulation of alpha 4-tk RN A in the absence of the alpha 4-2 binding site. We conclude that (i) t he inhibition of transcription by ICP4 is affected by both orientation and distance of the binding site from the TATA box, (ii) the model th at best fits the available data is that ICP4 bound to DNA at an approp riate distance and orientation interacts with and precludes the transc ription complex from transcribing the gene, and (iii) at 8 h after inf ection, additional factors such as protein binding upstream of the TAT A box and/or posttranslational modification of ICP4 strengthen the rep ressive effect of ICP4 at the alpha 4-2 binding site.