Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase

Abstract

Herpes simplex virus-induced DNA polymerase purified by published methods was found to be contaminated with many other proteins, including virus structural proteins. Thus, DEAE-cellulose and phosphocellulose chromatography were used in combination with affinity chromatography to purify DNA polymerase from herpes simplex virus type 1-and type 2-infected cells. The purified enzyme retained unique features of the herpesvirus-induced DNA polymerase, including a requirement for high salt concentrations for maximal activity, a sensitivity to low phosphonoacetate concentrations, and the capacity to be neutralized by rabbit antiserum to herpesvirus-infected cells. By polyacrylamide gel electrophoresis, the purified DNA polymerase was associated with a virus-induced polypeptide of about 150,000 molecular weight.

Nonstructural proteins of herpes simplex virus. II. Major virus-specific DNa-binding protein

“…The cells were pelleted by low-speed centrifugation, washed in Tris buffer (pH 7.5), and suspended in extraction buffer (20 mM Tris-hydrochloride [pH 7.5] and 0.5 mM dithiothreitol) at a cell concentration of 1 x 107 to 3 x 107/ml. The cells were then subjected to ultrasonic disruption and extracted with high salt as described previously (15). The extract was dialyzed ovemight against several changes of DE buffer (50 mM Tris-hydrochloride [pH 7.5], 0.5 mM dithiothreitol, 0.2% Nonidet P40, and 20% glycerol).…”

“…DNA polymerase assays. DNA polymerase assays and antiserum neutralization experiments were done as described previously (15).…”

“…We have previously associated a polypeptide of 150,000 molecular weight with the virus-specific DNA polymerase (15). A protein of about 44,000 molecular weight was associated with the virus-specific thymidine kinase (7), and a protein of 90,000 molecular weight was associated with the virus-induced alkaline exonuclease (21).…”

DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

“…The product of the UL42 gene (UL42) is one of seven proteins, required to replicate plasmids containing an HSV origin of replication (Wu et al, 1988), and is essential for viral DNA replication in vivo (Marchetti et al, 1988;Schaffer et al, 1973). Initially, UL42 was characterized as a phosphorylated 65-kDa double-stranded (ds)DNA binding protein being associated with purified HSV pol from serotype 1-and 2-infected cells (Gallo et al, 1988(Gallo et al, , 1989Marsden et al, 1987;Powell and Purifoy, 1977;Vaughan et al, 1985). From the open reading frame of the UL42 gene of HSV-1 strain 17, only a molecular weight of 51, 156 can be predicted .…”

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

“…The herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) is essential for viral replication and is an effective target for antiviral drug therapy (3,4,7,27). Recent work from a variety of laboratories is aimed at understanding the relationship between the structure of the viral pol and its enzyme activity.…”