The use of beta-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses.
The expression of Herpes Simplex Virus 1 (HSV-1) glycoprotein C (gC), a well defined herpesvirus late gene, was studied by linking the promoter-regulatory region of this gene to the coding sequences for the bacterial enzyme, beta-galactosidase (beta-gal). A chimeric gene, containing the beta-gal gene under the control of gC sequences from -1350 to +30 relative to the mRNA start site, was inserted by homologous recombination into the thymidine kinase (TK) locus of the HSV-1 genome. Selection of the TK- recombinant virus by plaque assay was facilitated by addition of a beta-gal indicator to the agarose overlay. Recombinant virus containing the gC promoter-beta-gal chimeric gene faithfully expressed beta-gal as a viral late gene, as shown by the absence of beta-gal expression when viral DNA replication was inhibited with phosphonoacetic acid. In contrast, the inhibition of viral DNA replication had no effect on the expression of beta-gal when the beta-gal gene was under the control of the early HSV-1 TK promoter in a separate recombinant virus. Analysis of recombinant viruses containing 5' to 3' deletions in the gC regulatory region revealed no apparent difference in beta-gal expression as deletions extended from -1350 to -109 base-pairs (bp) before the RNA start site, demonstrating that sequences between -109 and +30 are sufficient for regulated gC expression in the viral genome. Analysis of the mRNA made by these recombinant viruses confirmed the results of the beta-gal assays, and demonstrated that the transcriptional start sites of the gC promoter-beta-gal chimeric genes were the same as the start site of the gC gene.
Weir
J P
JP
Department of Microbiology, University of Tennessee, Knoxville 37996-0845.
Narayanan
P R
PR
D000818:Animals; D001483:Base Sequence; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005814:Genes, Viral; D005819:Genetic Markers; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D018139:Simplexvirus / Q000235:genetics*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
D005819:Genetic Markers; D014759:Viral Envelope Proteins; C038535:glycoprotein gC, herpes simplex virus type 1; D005696:Galactosidases; D001616:beta-Galactosidase