The herpes simplex virus (HSV) genome contains both cis-and transacting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oris and one copy of OriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the transacting functions required for the replication of plasmids containing oris or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode transacting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either onis or ofiL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.

The HSV-1 immediate early (IE) protein ICP4 (alpha 4, IE175, Vmw175) is an oligomeric molecule which activates transcription of viral early genes, represses transcription of viral IE genes, and binds to specific sequences in certain viral promoters. The extent to which these functions are interrelated has not been fully established. We have expressed truncated portions of the ICP4 gene in E. coli as trpE fusion proteins. DNA-binding studies with these hybrid proteins revealed that ICP4 residues 262 to 490 are sufficient for sequence-specific DNA-binding. DNA-binding was not detected with polypeptides extending from residue 262 to 464 or from residue 306 to 490. Multiple bands of protein-DNA complexes observed in gel mobility shift assays indicate that residues 262 to 490 may also contribute to the oligomerization of ICP4.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PRV), varicella-zoster virus (VZV), and equine herpesvirus 1 (EHV-1) are all classified as Alphaherpesvirinae. Each of these five viruses encodes an essential immediate-early (IE) regulatory protein referred to as HSV-1 1CP4, HSV-2 ICP4, PRV IE180, VZV 0RF62 protein, and EHV-1 TEl, respectively. These five proteins share extensive homology with each other in domains referred to as regions 2 and 4. The HSV-1 ICP4 region 2 domain contains residues that are required for the DNA-binding capability of ICP4. In this report, we describe the expression of region 2 domains from the ICP4, IE180, and 0RF62 genes as fusion proteins in Escherichia coli. DNA-binding assays revealed that each of these region 2 fusion proteins binds to a sequence that overlaps the transcription start site in the promoter for the gene encoding the corresponding protein. Each of the sites with high affinity for one or more of these fusion proteins contains the sequence 5'-ATCGT-3'. This sequence spans the mRNA cap site in the HSV-2 ICP4 gene promoter and is immediately upstream from the transcription start site in the EHV-1 IEl gene. These results suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene.