The use of β-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses

摘要

The expression of Herpes Simplex Virus 1 (HSV-l) 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, β-galactosidase (β-gal). A chimeric gene, containing the β-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 β-gal indicator to the agarose overlay. Recombinant virus containing the gC promoter-β-gal chimeric gene faithfully expressed β-gal as a viral late gene, as shown by the absence of β-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 β-gal when the β-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 β-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 β-gal assays, and demonstrated that the transcriptional start sites of the gC promoter-β-gal chimeric genes were the same as the start site of the gC gene.