Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity.
A cloned herpes simplex virus type 1 DNA polymerase gene which is biologically functional was inserted into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. pol-specific RNA synthesized in vitro will direct the synthesis of a 140-kilodalton polypeptide in rabbit reticulocyte lysates. RNAs prepared from pol templates linearized at internal restriction sites specified deleted polypeptides with sizes consistent with colinearity of the pol gene and the 140-kilodalton primary translation product. The in vitro translated pol gene product was enzymatically active, with salt resistance and sensitivity to acyclovir triphosphate, similar to the enzyme activity in crude extracts of herpes simplex virus type 1-infected Vero cells. An in-frame deletion of 78 residues (amino acids residues 881 to 959) was introduced into the expression vectors to investigate the function of a region of the polypeptide (amino acids residues 881 to 895) which is conserved in nine other DNA polymerases. In a complementation assay, this mutation abolished biological activity as well as the enzymatic activity of the in vitro translated product. A BAL31 mutation deleting the upstream open reading frame of pol had no effect on biological activity in a complementation assay but was found to increase the efficiency of in vitro translation of pol RNA. Two amino-terminal deletions of 27 and 67 residues were found to greatly enhance the enzymatic activity of the in vitro translated product, while all carboxy-terminal deletions examined (the smallest being 164 residues) abolished in vitro enzymatic activity. Expression of the 67-residue amino-terminal deleted pol gene in Escherichia coli, using a bacteriophage T7-based system, resulted in accumulation of large amounts of an insoluble fusion protein. An antiserum prepared against this fusion protein precipitated the 140-kilodalton DNA polymerase from herpes simplex virus type 1-infected cell lysates.
Dorsky
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Charles A. Dana Research Institute, Boston, Massachusetts.
Crumpacker
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D000818:Animals; D002460:Cell Line; D004256:DNA Polymerase I / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005816:Genetic Complementation Test; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014709:Vero Cells
D004256:DNA Polymerase I