title: The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts.
abstract: The isolation of viruses with mutations in essential genes requires that they be propagated in cells expressing the wild-type proteins. This has been a particularly challenging problem for studying mutations in the human cytomegalovirus (HCMV) immediate early (IE) gene, IE2 86. In the past, we tried a number of approaches to derive human fibroblasts expressing wild-type IE2 86, but were unable to maintain expression of a fully functional protein. To overcome this obstacle, we developed a strategy whereby recombinant baculoviruses were used as vectors for the expression of HCMV IE proteins in primary human fibroblasts (FFs). The IE2 86 and IE1 72 cDNAs, as well as the genomic fragment of the UL122-123 region under the control of a chicken actin promoter, were introduced into the baculovirus genome by site-specific transposition in Escherichia coli. Recombinant "bacmid" DNAs were then transfected into Sf9 cells to generate recombinant baculoviruses. FFs infected at high m.o.i. with these baculoviruses expressed high levels of the HCMV protein for at least 1 week, as determined by immunofluorescence assays and Western blots. Moreover, the IE2 86 protein was found to be fully functional with respect to its ability to activate the HCMV UL112-113 early promoter. Recombinant baculoviruses expressing IE1 72 were also able to efficiently complement HCMV ie1 mutants. These data demonstrate the potential of using recombinant baculoviruses as vectors for the expression of toxic viral genes in human cells and for subsequent isolation of mutant HCMV lacking these essential genes.
authors.affiliation: Molecular Biology Section and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0366, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Dwarakanath
authors.forename: C L
authors.initials: CL
authors.lastname: Clark
authors.forename: A K
authors.initials: AK
authors.lastname: McElroy
authors.forename: D H
authors.initials: DH
authors.lastname: Spector
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism* / Q000821:virology*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D014162:Transfection
chemical_list: D017874:Immediate-Early Proteins