ABSTRACT
An in vitro system using nuclei from parvovirus H-1-infected cells was used to characterize the influence of inhibitors of mammalian DNA polymerases on viral DNA synthesis. The experiments tested the effects of aphidicolin, which is highly specific for DNA polymerase alpha, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order gamma greater than beta greater than alpha. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase alpha and a ddttp-sensitive enzyme are required for viral DNA synthesis. This was seen more clearly in kinetic measurements, which indicated an initial period of rapid DNA synthesis with the participation of polymerase alpha, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTP-sensitive enzyme, probably polymerase gamma. One interpretation of the results is that polymerase alpha functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase gamma serves to convert the displaced single strands back to double-strand replicative form.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/metabolism , Parvoviridae/enzymology , Viral Proteins/metabolism , Aphidicolin , DNA, Viral/biosynthesis , Dideoxynucleotides , Diterpenes/pharmacology , Nucleic Acid Synthesis Inhibitors , Thymine Nucleotides/pharmacology , Viral Proteins/antagonists & inhibitors , Virus ReplicationABSTRACT
The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma.
Subject(s)
DNA Polymerase III/metabolism , DNA Replication , DNA, Viral/genetics , DNA-Directed DNA Polymerase/metabolism , Parvoviridae/genetics , Cell Line , Female , Humans , Kidney/embryology , Molecular Weight , Parvoviridae/enzymology , Placenta/enzymology , Pregnancy , Templates, Genetic , Virus ReplicationABSTRACT
We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl(2) precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase beta had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases alpha or gamma. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm(2) in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Parvoviridae/enzymology , Parvovirus/enzymology , Animals , Cattle , Cell Line , Centrifugation, Density Gradient , Parvoviridae/growth & development , Parvoviridae/isolation & purification , Parvovirus/growth & development , Parvovirus/isolation & purification , Virus ReplicationSubject(s)
Adenoviridae/genetics , Parvoviridae/genetics , RNA, Viral/genetics , Transcription, Genetic , Cell Line , Cell Nucleus/metabolism , DNA, Viral/genetics , DNA-Directed RNA Polymerases/metabolism , Nucleic Acid Hybridization , Parvoviridae/enzymology , RNA/biosynthesis , RNA, Viral/biosynthesis , Time Factors , Uridine Triphosphate/metabolismSubject(s)
DNA-Directed DNA Polymerase , Adenoviridae/enzymology , Animals , Cell Differentiation , DNA Polymerase I/metabolism , DNA Polymerase II/metabolism , DNA Polymerase III/metabolism , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Ethylmaleimide/pharmacology , Hepatitis B virus/enzymology , Herpesviridae/enzymology , Hydroxymercuribenzoates/pharmacology , Mitochondria/enzymology , Molecular Weight , Papillomaviridae/enzymology , Parvoviridae/enzymology , Polyomaviridae , Species Specificity , Templates, GeneticABSTRACT
Purified preparations of the parvovirus, Kilham rat virus, have associated with them a protein with DNA polymerase activity. The enzyme has been separated from the other two or three viral proteins and purified 63-fold. The viral associated enzyme was found in a single peak of DNA polymerase activity after chromatography on DEAE-cellulose, DNA-cellulose, and phosphocellulose columns. It shares some properties in common with the host cellular DNA polymerases, described in the preceding paper (Salzman, L.A., and McKerlie, L. (1975) J. Biol. Chem. 250, 5589-5595), but also has some important distinguishing characteristics. The Kilham rat virus-associated DNA polymerase has increased enzyme activity in the presence of 0.02 M KCl and has a strong preference for a synthetic DNA polymer containing deoxyadenylate and deoxythymidylate. The enzyme has a molecular weight of approximately 75,000 plus or minus 3,000 and appears to contain endonuclease activity.
