No abstract

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 six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunitsubunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.The initiation of DNA replication in lower eukaryotes is similar to that observed in bacteria in that it occurs at well defined origins of DNA replication that are recognized by specific initiator proteins (1-5). Saccharomyces cerevisiae origins of DNA replication are short, ϳ100-base pair (bp) 1 segments that consist of two essential regions: the A domain, which contains a conserved 11-bp consensus sequence (ACS) that is required for origin function, and the B domain, which contains several stimulatory elements (6, 7). The ACS is recognized specifically by the S. cerevisiae initiator protein, the origin recognition complex (ORC) (8, 9). S. cerevisiae ORC (ScORC) is comprised of six proteins (Orc1-6), each of which is essential for viability in S. cerevisiae (10 -13). A number of lines of evidence indicate that ScORC is required for initiation of DNA replication in vivo and functions in part to recruit other initiation factors, such as Cdc6 and the minichromosome maintenance proteins, to replication origins (13-15).Homologues of ORC subunits have been identified in a variety of other eukaryotic species (2, 16), and ORC has been purified from the fission yeast Schizosaccharomyces pombe and the metazoans Drosophila melanogaster and Xenopus laevis (17)(18)(19). Genetic and biochemical data indicate that specific ORC subunits from t...

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, onis and orML. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on onis, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described (14), and the recombinant plasmid was used to generate the recombinant baculovirus AcNPV/UL9 as described (15,16).Antisera. Decapeptides corresponding to the predicted carboxyl-terminal amino acid sequence ofHSV-1 genes UL5, UL8, UL9, and UL52 were purchased from Biosearch (San Rafael, CA) and were covalently coupled to keyhole limpet hemocyanin. Approximately 0.5 mg of coupled peptide was used to immunize rabbits biweekly for a total of three injections. Sera were collected at biweekly intervals beginning one month after the first injection. A complete characterization of these sera will be published elsewhere.Preparation of Baculovirus-Infected Cell Extract. Fifteen 150-cm2 flasks of nearly confluent SF9 cells were infected with the recombinant virus AcNPV/UL9 at a multiplicity of infection of 10-20 plaque-forming units per cell. After 54 hr at 28°C, the cells were dislodged from the flasks by shaking and were washed with phosphate-buffered saline. NucleiAbbreviations: HSV, herpes simplex virus; AcNPV, Autographa californica nuclear polyhedrosis virus.