Doctoral Dissertations

Date of Award

5-1990

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Barry Rouse

Committee Members

Jeff Becker, Leon Potgieter, Jerry Weir

Abstract

Expression of the Herpes Simplex Virus Type 1 (HSV-1) genes is a well coordinated process which results in the production of three groups of proteins according to their temporal order of synthesis; immediate early (α), early (β), and late (γ) genes (Honess and Roizman, 1974, 1975). To investigate the cis-acting sequence elements which are involved in the regulation of HSV-1 late gene expression, we have constructed recombinant herpesviruses expressing the bacterial enzyme β-galactosidase (β-gal) from the gH promoter. A chimeric gene, containing the β-gal gene under the control of gH sequences from -1400 to +198 relative to the start of transcription, was inserted into the viral genome at the thymidine kinase locus by homologous recombination. This recombinant virus faithfully expressed β-galactosidase as a late gene, as determined by its strict requirement for viral DNA replication. Deletions from -1400 to -89 established an upstream boundary for sequences necessary for accurate regulation of expression from the gH promoter. In addition, specific site-directed mutations were created in the region spanning from -76 to -26, which includes the Thymidine Kinase (TK) polyadenylation signal, AATAAAAA, a CCAAT-like element, and the gH TATA-like element, AATAAAA, and the expression of β-galactosidase was determined in infected cells. Analysis of these recombinant viruses revealed the requirement of a TATA box for full expression. Further, mRNA synthesis from the recombinants demonstrated that the start of transcription from the gH promoter-β-gal genes was the same as the start of the authentic gH gene. In addition, the gH TATA box element was specifically mutagenized to substitute the TATA box elements of immediate-early, early, and other late viral genes for the gH TATA box. Analysis of these recombinant viruses revealed that each mutated promoter expressed β-gal as a late viral gene and directed correct transcription initiation, as determined by primer extension experiments. A series of 3' to 5' deletions in the 5' noncoding region of the gH gene established the 3' boundary of the gH promoter at position +46, relative to the start of gH transcription. Subsequently, eight linker scanning mutations were constructed in the gH promoter from -32 to +43. Analysis of β-galactosidase expression and mRNA synthesis from these recombinant viruses demonstrated the requirement not only for the TATA element, but sequences at the transcriptional start site of gH. These findings suggest that HSV-1 late promoters may consist of multiple sequence elements that include a TATA element and a sequence element at the start of transcription. The role of each of these elements in directing transcription initiation and in determining the magnitude of expression were analyzed by constructing hybrid late promoters using sequences from the glycoprotein C and gH promoters.

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