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We have performed a functional analysis of DNA sequences upstream from the gene for IE mRNA3 of herpes simplex virus type 1. Nucleotide sequences involved in initiation and positive regulation of transcription have been defined by construction of specific deletions in vitro. Transcription was assayed in vivo by microinjection into Xenopus oocytes, or by introduction of plasmid DNA into tissue culture cells and measurement of transient expression. Three functional promoter elements have been defined: i) Sequences between -16 and -37 which are not essential for transcription but are required for accurate initiation. ii) Proximal promoter sequences which are sufficient for transcription initiation in the absence of upstream sequences. iii) Far-upstream promoter sequences (more than 108bp upstream) which increase transcription in oocytes, and contain positive regulatory sequences (-174 to -331) which respond strongly to a factor in the virus inoculum.

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The possible identity of the herpes simplex virus type 1 (HSV-1) 65K (65,000-Mr) virion protein which stimulates transcription from immediate-early genes with the HSV-1 65K DNA-binding protein was investigated. The two proteins were found to be distinct by the three separate criteria of immunological reactivity, tryptic peptide fingerprinting, and mobility in two-dimensional gels. Using HSV-1/HSV-2 intertypic recombinants and a serotype-specific antiserum, we located the gene encoding the 65K DNA-binding protein between coordinates 0.574 and 0.682 on the HSV-1 genome. The protein is posttranslationally modified by phosphorylation. In crude extracts of HSV-1-infected cells the 65K trans-inducing protein did not detectably bind to double-stranded calf thymus DNA under the conditions of our assay. Expression of herpes simplex virus (HSV) genes is temporally regulated and is considered to occur in at least three phases termed immediate-early (IE), early, and late or (x, P, and-y (13, 27, 28, 31, 50). IE genes are expressed in the absence of de novo protein synthesis (27) but their expression is stimulated by a component of the virus particle (5, 42). This trans-inducing factor was identified as polypeptide Vmw65 (11) and is designated 65KTIF in the present study. The mechanism by which 65KTIF stimulates transcription from IE genes is not understood; however, one possibility is that it interacts with some IE-gene-regulatory DNA sequence. Interestingly, a responder element located several hundred bases upstream of the IE mRNA 5' terminus has been identified and corresponds to the consensus sequence TAATGARATTC (R is purine) (10, 35, 44, 53). Previously, we identified a major DNA-binding protein in HSV type 1 (HSV-1)-infected cells with an apparent Mr of ca. 62,000 (6). This DNA-binding protein and 65KTIF have the same electrophoretic mobilities in sodium dodecyl sulfate-5 to 12.5 polyacrylamide gels, and so we designated the DNA-binding protein 65KDBP. It seemed important to establish whether 65KDBp and 65KTIF were one and the same protein and whether 65KTIF can bind to DNA. This study addresses these questions. We characterized the two proteins and show that 65KDBp and 65KTIF are distinct and that 65KTIF does not detectably bind to DNA under the conditions of our assay. MATERIALS AND METHODS Cells. BHK21 clone 13 cells (34) were used throughout. Viruses. HSV-1 strain 17syn+ (9) and HSV-2 strain HG52 (51) were used in this study. The isolation of HSV-1/HSV-2 intertypic recombinants and the determination of their genome structures have been previously reported (12, 15, 37, 46, 54). Radioactive labeling. Confluent monolayers in 50-mmdiameter dishes or roller bottles were infected at a multiplicity of infection of 5 to 20 PFU of HSV-1 per cell. After 1 h, * Corresponding author.