Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction.
The pseudorabies virus (PRV) genes encoding the two subunits of the DNA polymerase were located on the genome by hybridization to their herpes simplex virus type 1 (HSV-1) homologs, pol and UL42, and subsequently were sequenced. Like the HSV-1 homologs, in vitro translation products of the PRV gene encoding the catalytic subunit (pol) possessed activity in the absence of the Pol accessory protein (PAP). However, the PRV PAP stimulated the activity of Pol fourfold in the presence of 150 mM KCl, using an activated calf thymus DNA template. The stimulation of Pol activity by PAP under high-salt conditions and the inhibition of Pol activity by PAP when assayed in low salt (0 mM KCl) together were used to determine the specificity with which PAP interacted with Pol. Despite functional similarity, HSV-1 UL42 and PRV PAP could neither stimulate the noncognate Pols at high salt nor inhibit them at low salt. Furthermore, a PRV Pol mutant lacking the 30 C-terminal amino acids retained basal Pol activity but could be neither stimulated nor inhibited by the PRV PAP. Sequence comparisons of the Pol proteins of the alphaherpesviruses reveal a conserved domain in the C terminus which terminates immediately before the last 41 residues of both PRV and HSV-1 proteins. These results indicate that the ability and specificity for interaction of the PRV Pol with PAP most likely resides predominantly in the extreme Pol C terminus.
Berthomme
H
H
Centre de Génétique Moléculaire et Cellulaire UMR 106, Centre National de la Recherche Scientifique, Université Claude Bernard Lyon I, Villeurbanne, France.
Monahan
S J
SJ
Parris
D S
DS
Jacquemont
B
B
Epstein
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005814:Genes, Viral; D015751:Genes, pol; D006564:Herpesviridae / Q000201:enzymology / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics; D011558:Herpesvirus 1, Suid / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D011189:Potassium Chloride / Q000494:pharmacology; D014176:Protein Biosynthesis; D011487:Protein Conformation; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells
D011189:Potassium Chloride; D004259:DNA-Directed DNA Polymerase