Nucleic Acids Research, 1988, Vol. 16, No. 21 10267-10282
© 1988
MOLECULAR BIOLOGY |
The use of ß-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses
Department of Microbiology, University of Tennessee Knoxville, TN 37996–0845, USA 1Department of Immunology, Tuberculosis Research Center Madras 600 031, India
* To whom correspondence should be addressed
Received June 7, 1988. Accepted September 27, 1988.
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, ß-galactosidase (ß-gal). A chimeric gene, containing the ß-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 ß-gal indicator to the agarose overlay. Recombinant virus containing the gC promoter-ß-gal chimeric gene faithfully expressed ß-gal as a viral late gene, as shown by the absence of ß-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 ß-gal when the ß-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 ß-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 ß-gal assays, and demonstrated that the transcriptional start sites of the gC promoter-ß-gal chimeric genes were the same as the start site of the gC gene.