Replication of parvoviral DNA. I. Characterization of a nuclear lysate system

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 a, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order y > , > a. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase a 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 a, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTPsensitive enzyme, probably polymerase y. One interpretation of the results is that polymerase a functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase-y serves to convert the displaced single strands back to double-strand replicative form.

Section: Discussionmentioning

confidence: 75%

DNA polymerase requirements for parvovirus H-1 DNA replication in vitro

Kollek¹,

Tseng²,

Goulian³

1982

“…The absence of Okazaki fragments during parvovirus H-1 replication (39) and the ability of the hairpins to serve as self-primers obviate the need for a DNA primase which is associated with polymerase a. Parvovirus replication is more similar to leading-strand DNA synthesis, which appears to be carried out by polymerase 8 (27,35). Studies showing polymerase a as the replicating enzyme for BPV (28,30), rat virus (16), and parvovirus H-1 (21) were based on sensitivity to aphidicolin and resistance to dideoxy-TTP, but both polymerases a and 8 share these properties.…”

Section: Discussionmentioning

confidence: 99%

Interaction of virally coded protein and a cell cycle-regulated cellular protein with the bovine parvovirus left terminus ori

Metcalf

Bates

Lederman

1990

Self Cite

Replication of parvoviruses requires cis signals located in terminal palindromes that function as origins of replication in conjunction with transacting viral and cellular proteins. A gel retardation assay was used to identify proteins in crude nuclear extracts of bovine parvovirus (BPV)-infected bovine fetal lung cells that interact with the hairpinned left end (3' OH terminus of the viral minus strand in the flop conformation) of BPV. Three specific DNA-protein complexes formed. One complex was shown to involve a BPV structural protein(s) by inhibiting its formation when antiserum specific for these BPV proteins was used. By specific competition with serum containing antibodies against the BPV nonstructural proteins, a second complex was shown to involve a BPV nonstructural protein. A third complex contained protein of cellular origin and was also formed with extracts of uninfected bovine fetal lung cells. DNA competition assays suggest that the viral proteins do not bind to the right hairpin, which differs in sequence and secondary structure from the left terminus, or to a BPV terminus that lacks the first 52 nucleotides, preventing formation of the stem of the hairpin. The cellular protein is regulated in a cell cycle-dependent fashion, with its binding activity increased in uninfected, actively dividing cells compared with contact-inhibited cells. Since autonomous parvovirus replication requires an S-phase factor for progeny formation, the terminal binding protein demonstrated here is a candidate for this factor.

“…However, these results do not rule out the possibility that both pola and poly activities are necessary for the production of parental RF. Previous studies with subcellular systems to study parvoviral DNA replication have focused on the in vitro polymerase requirements for later stages in the replication cycle (9,15,17). Data obtained with partially purified (3; Robertson et al, Fed.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, partially purified DNA pola (3) and purified pola reconstituted with specific protein cofactors (but not purified polot alone) (16) utilized the parvoviral DNA template. Studies with specific inhibitors indicated that in vitro polot functioned in parvoviral DNA replication in both isolated nuclei (9) and a nuclear lysate system (15). Moreover, in vivo levels of DNA polcx increased during the parvoviral infection cycle (13).…”

mentioning

confidence: 99%

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection

Robertson¹,

Stout²,

Bates³

1984

Self Cite

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pola, pol3, or poly, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA polot, to assay for the requirement of pola activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 pFM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 ,uM aphidicolin, whereas lower concentrations (0.1 and 0.01 FM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA polot is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA poly in the first step, nor do they rule out a role for polot or pol-y in later stages of the replication cycle.