The herpes simplex virus type I DNA polymerase. Polypeptide structure and antigenic domains.
Polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the herpes simplex virus type I (HSV-1) DNA polymerase of strain Angelotti were generated. Each of the five different rabbit antisera reacted specifically with a viral 132 +/- 5-kDa polypeptide as shown by immunoblot analysis. Enzyme binding and inhibition studies revealed that antibodies raised to the central and the C-terminal domains of the protein inhibited the polymerizing activity by 70-90%, respectively, which is well in line with the proposed site of the catalytic center of the enzyme and with the possible involvement of these polypeptide chains in DNA-protein interactions. In agreement with this, antibodies directed towards the N-terminal domain bound to the enzyme without effecting the enzymatic activity. The strong binding but low inhibitory properties of antibodies directed to the polypeptide region between residues 1072 and 1146 confirms previous suggestions that these C-terminal sequences, which share no homology to the Epstein-Barr virus DNA polymerase, are less likely involved in the building of the polymerase catalytic site. Antibodies, raised to the very C terminus of the polymerase (EX3), were successfully used to identify a single 132 +/- 5-kDa polypeptide, which coeluted with the HSV DNA polymerase activity during DEAE-cellulose chromatography, and were further shown to precipitate a major viral polypeptide of identical size. From the presented data it can be concluded that the native enzyme consists of a single polypeptide with a size predicted from the long open reading frame of the HSV-1 DNA polymerase gene.
Weisshart
K
K
Institut für Virusforschung, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany.
Knopf
C W
CW
D000914:Antibodies, Viral / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000276:immunology; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D010957:Plasmids; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014169:Transformation, Bacterial
D000914:Antibodies, Viral; D000936:Antigen-Antibody Complex; D000939:Epitopes; D004256:DNA Polymerase I