title: Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells.
abstract: 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.
authors.affiliation: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
authors.forename: Victoria
authors.initials: V
authors.lastname: Ellsmore
authors.affiliation: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
authors.forename: G Gordon
authors.initials: GG
authors.lastname: Reid
authors.affiliation: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
authors.forename: Nigel D
authors.initials: ND
authors.lastname: Stow
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D009693:Nucleic Acid Hybridization; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D014764:Viral Proteins