The herpes simplex virus type I DNA polymerase

Abstract

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 rexted 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 f 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.The herpes simplex virus type I (HSV-1) DNA polymerase represents an attractive model enzyme for studies of eukaryotic DNA polymerases. Like the M: polymerase, the key enzyme of cellular DNA synthesis, the HSV DNA polymerase is of central importance for successful viral replication. The viral enzyme has been involved in the initiating events of the gene amplification processes [l], has been shown to play a key role in viral mutagenesis [2] and is one of the major target enzymes of modern antiviral chemotherapy. The analysis of HSV mutants, which induce a drug-resistant and/or a temperature-sensitive polymerase, by marker rescue as well as by sequencing studies has allowed us to identify the gene encoding the viral enzyme (Fig.

Conformational changes induced in herpes simplex virus DNA polymerase upon DNA binding.

“…This region also contains a highly conserved aspartic residue that is thought to be involved in catalysis in diverse polymerases (15,16 DNA once it is bound to the cleft in the C-terminal domain (5), for which there is now x-ray crystallographic evidence (L. Beese and T. Steitz, personal communication). In this regard, it is interesting that residues -1080-1140 are predicted to be hydrophilic and flexible (23,39).…”

“…The catalytic subunit of HSV Pol was purified from recombinant baculovirus BP58-infected Sf9 cells (22) by a procedure to be described elsewhere (K.W., A.A.K., C. B. C. Hwang, and D.M.C., unpublished work). Antibod-ies EX6, EX1051, EX3, BGG4, and PP5 have been described (23,24). Activated calf thymus DNA (Sigma) was purified (25) 40 ,ug/ml for 2 min on ice, whereas V8 protease digestion was terminated by leupeptin at 0.5 1kg/ml.…”

A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant.

“…The resulting supernatants or reticulocyte lysates were subjected to immunoprecipitation as described (33) using antisera specifically reacting with HSV-1 TK (kindly provided by W. C. Summers, Yale University, New Haven) or the C-terminal portion of HSV-1 Pol (EX3; kindly provided by K. Weisshart and C. Knopf, Deutsches Krebsforschungszentrum, Heidelberg; ref. 34). 35S-labeled polypeptides were separated on SDS/polyacrylamide gels and visualized by fluorography with Amplify (Amersham) by using preflashed film.…”

Epitope Mapping and Functional Characterization of Monoclonal Antibodies Specific for Herpes Simplex Virus Type I DNA Polymerase

“…E. Streck and C. Franz, respectively. DK.FZ, Heidelberg, Germany) were prepared as de scribed elsewhere [12]. Rabbit monospecific anti-UL29 IgG was a kind gift from W. Ruyechan (State University of New York, Buffalo, N.Y., USA).…”

“…As tools for the structural and functional dissection of the HSV core replication complex, we have previously generated polyclonal antibodies against 12 partly overlapCharles W. Knopf ping subdomains of HSV pol from strain Angelotti (ANG), encompassing the complete protein sequence [12,13]. Antibodies directed against the central domain (resi dues 597-685) were found to be very effective in binding HSV pol and in neutralizing both 3'-5' exonuclease and polymerization activities [2].…”