An analysis of the requirements for human cytomegalovirus oriLyt-dependent DNA synthesis in the presence of the herpes simplex virus type 1 replication fork proteins.

Activation of the human cytomegalovirus (HCMV) origin of replication (oriLyt) was previously demonstrated in transient transfection assays in permissive human fetal fibroblasts and nonpermissive Vero cells, and shown to require six viral proteins that function at the replication fork plus a number of HCMV products that perform auxiliary roles. The six replication fork proteins could be substituted by their Epstein-Barr virus homologues. In this paper we demonstrate that the corresponding herpes simplex virus type 1 replication fork proteins can similarly replace those of HCMV in Vero cells. Under these conditions the essential auxiliary functions were mapped to two plasmids: pSVH (containing the major immediate-early locus) and pZP8 (spanning genes UL32-UL38). Mutants of pSVH and pZP8 and cloned cDNAs encoding the IE1-p72 and IE2-p86 proteins were tested for their ability to support DNA synthesis. The results showed that IE2-p86 was necessary for activation of the origin, and that the UL37x1 and IE1-p72 products exerted strong stimulatory effects. In contrast to the previous work, omission of the UL84 protein had no effect upon oriLyt-dependent DNA synthesis.

Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells.

Human cytomegalovirus (HCMV) displays an exceptionally restricted host range in tissue culture with human fibroblasts being the principal fully permissive system. Nevertheless, immediate early (IE) proteins are expressed following infection of many non-permissive cell types of human, simian and murine origin, and viral origin-dependent DNA synthesis has been reconstituted by transfection of plasmids into Vero cells, a non-permissive line from African green monkey. We have examined the accumulation of HCMV strain AD169 DNA, and the replication of transfected HCMV origin-containing plasmids, in infected Vero and human embryonic kidney 293 cells, which were previously reported to express the major IE protein in a small proportion of infected cells but to be non-permissive for viral DNA synthesis. In Vero cells accumulation of origin-containing plasmid but not viral DNA occurred, whilst in 293 cells both DNAs accumulated. Immunofluorescence experiments indicated that following infection with 3 p.f.u. per cell, a small fraction of both cell types expressed the UL44 DNA replication protein. Neither cell line, however, supported the generation of infectious progeny virus. These results suggest that IE proteins expressed in Vero and 293 cells can induce the synthesis of early proteins capable of functioning in viral DNA replication, but there is a failure in later events on the pathway to infectious virus production. This provides further support for transfected Vero cells being a valid system in which to study HCMV DNA synthesis, and suggests that 293 cells may also prove useful in similar experiments.