EVIDENCE THAT THE UL84 GENE-PRODUCT OF HUMAN CYTOMEGALOVIRUS IS ESSENTIAL FOR PROMOTING ORILYT-DEPENDENT DNA-REPLICATION AND FORMATION OF REPLICATION COMPARTMENTS IN COTRANSFECTION ASSAYS

The protein products of 11 viral genomic loci cooperate in a transient cotransfection assay to mediate lytic-phase DNA replication of oriLyt , the human cytomegalovirus (HCMV) origin of replication, Six of these genes have homology with the well-characterized herpes simplex virus replication genes and encode core replication machinery proteins that are typically essential for DNA synthesis, The remaining five HCMV gen e loci, initially referred to as auxiliary components, include several known immediate early (IE) transcriptional regulatory proteins as wel l as genes encoding functionally uncharacterized polypeptides. Some or all of the auxiliary components may be necessary in trans to replicat e the HCMV oriLyt only because they are required for efficient express ion or transactivation of the native early promoters and 3' processing elements included in the genomic clones, Therefore, we reassessed the requirements for the auxiliary components by adding constitutive hete rologous promoters and control signals to the coding regions and carry ing out transient DpnI replication assays in cotransfected Vero cells, The results revealed that in the presence of the UL69 posttranscripti onal activator and the remaining auxiliary polypeptides, UL84 was the only auxiliary component that could not be omitted to obtain oriLyt-de pendent DNA replication, Nevertheless, in human diploid fibroblasts, s ome additional auxiliary loci as well as UL84 were critical, There was also an obligatory requirement for UL84, in cooperation with two othe r auxiliary factors, UL112-113 and IE2, and the core machinery, to con stitute the minimal HCMV proteins necessary to direct oriLyt-dependent DNA amplification, However ever, the Epstein-Barr virus fore replicat ion genes could substitute for the HCMV core genes, and in these circu mstances, UL84 alone directed amplification of HCMV oriLyt. Moreover, there was also an absolute requirement for UL84 along with the core an d other auxiliary factors for the formation of intranuclear replicatio n compartments as assayed by immunofluorescence in transient DNA cotra nsfection assays, These compartments were typical of those associated with active viral DNA replication in HCMV-infected cells, they incorpo rated pulse-labeled bromodeoxyuridine, and their formation was both ph osphonoacetic acid sensitive and oriLyt dependent, These results demon strate that UL84 is obligatory for both intranuclear replication compa rtment formation and origin-dependent DNA amplification and suggest th at it is a keg viral component in promoting the initiation of HCMV ori Lyt-directed DNA replication.