Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates

Abstract

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-I) promoter. Genes controlled by the GAL-I promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose or galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1-140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited >90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.

In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme

“…Recently, the construction of a Saccharomyces cerevisiae expression system which produces an active form of the HSV-1 (strain 17) DNA pol was reported (14). In the present study, a specific point mutation was introduced into the yeast-HSV-1 pol expression vector, resulting in a predicted change in the reported protein sequence (13,28) at residue 815, converting asparagine to serine.…”

“…The original vector, pMH202 (14), was modified to contain HSV-1 (strain KOS) pol sequences, yielding pRC203 ( Fig. 1), so as to be consistent with ongoing biochemical studies with HSV-1 (KOS) in our laboratories.…”

“…Summary of the HSV-1 DNA pol-yeast expression vectors used in this study. GAL1, CYCl, URA3, BLA, and 2,um (2,u) are as previously described (14) and denote, respectively, the S. cerevisiae galactokinase-1 gene promoter, a synthetic oligonucleotide transcription termination sequence, a selectable marker for growth in S. cerevisiae, a selectable marker for growth in E. coli, and the 2,m S. cerevisiae replication sequence. Restriction endonuclease sites are MluI, SstI, PstI, KpnI, XbaI (X), and Hindlll (H).…”

“…Thick bars are HSV-1 (strain 17) sequences; thin bars are HSV-1 (KOS) sequences. (a) The construction of pMH202 is described in detail elsewhere (14). (b) pRC203 is derived from pMH202: the pol region from MluI to KpnI has been replaced by the analogous region of HSV-1 (KOS) DNA from pNN3, a plasmid constructed and generously supplied by M. D. Challberg (3).…”

Isolation and Characterization of theCandida albicans PFY1 Gene for Profilin

“…The sequence of this intron-less copy of the gene was verified by DNA sequencing. The 400 bp gene was extracted from the plasmid with Asp718I and BamHI and placed behind the GAL1 promoter in plasmid pMH101 (Haffey et al, 1988) cut with BamHI and HindIII. The ligation was accomplished by filling-in the Asp718I and HindIII sites and gave plasmid pDO3.…”