Isolation of intracellular replicative forms and progeny single strands of DNA from parvovirus KRV in sucrose gradients containing guanidine hydrochloride

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or a-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.

Section: Methodsmentioning

confidence: 99%

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

Pritchard¹,

Stout²,

Bates³

1981

“…Experiments with Escherichia coli or phage T4 DNA polymerases demonstrate that the 3' hairpin double-stranded DNA can serve as a self-primer for the synthesis of a complementary linear viral DNA strand (2,19). A double-stranded viral DNA molecule has been reported in infected cells with the properties of terminal hairpins and has been proposed as an intermediate in the replication of the viral genome (8,10,15,21). Because ofthe importance of the 3' terminus of the viral DNA in viral DNA replication and possibly in transcription, we have undertaken the sequence analysis of the 3'terminal nucleotides.…”

mentioning

confidence: 99%

Nucleotide sequence of the self-priming 3' terminus of the single-stranded DNA extracted from the parvovirus Kilham rat virus

Salzman¹,

Fabisch²

1979

The parvovirus genome is a linear, single-stranded DNA molecule with double-stranded hairpin termini. The 3' terminus can serve in vitro as a self-primer for the synthesis of a double-stranded viral DNA intermediate. We have sequenced the nucleotides in the 3' terminus and propose a model for the secondary structure of the terminus and the in vitro origin of replication for the complementary viral DNA strand.

“…BPV DNA from infected cells pulse-labeled at intervals (see Fig. 2B) was analyzed on sucrose gradients containing 4 M guanidine hydrochloride by the method of Lavelle and Li (7).…”

mentioning

confidence: 99%

“…Infectivity was determnined for parallel cultures by plaque assay on BFS cells. 7,753 (65)b 7,393 (72) 1.67 5,854 (73) 5,182 (80) aEnzyme preparations preincubated with antibody at 20°C for 10 min, then assayed in DNA polymerase a reaction mixture.…”

mentioning

confidence: 99%

Levels of cellular DNA polymerases in synchronized bovine paravovirus-infected cells

Pritchard¹,

Bates²,

Stout³

1978

Infection of synchronized bovine fetal spleen cells with bovine parvovirus results in changes in the levels and patterns of DNA polymerases a and y during the cell cycle. The pattern of DNA polymerase a activity closely paralleled viral DNA synthesis and the production of progeny virus, and levels of this enzyme were threefold greater than in mock-infected cells during the period of maximal viral DNA synthesis. DNA polymerase y activity remained slightly elevated during viral DNA replication. Levels and patterns of DNA polymerase ,B were similar in mockand virus-infected cells.