Replication Process of the Parvovirus H-1 II. Isolation and Characterization of H-1 Replicative Form DNA

Abstract

The Parvovirus H-1 replicates autonomously in hamster embryo cells. A DNA synthetic event, called HA-DNA synthesis, upon which subsequent viral RNA and viral hemagglutinin synthesis is dependent, is initiated in late S phase of the infected cell (18). It was postulated that HA-DNA represents parental viral replicative form DNA (RF DNA). This study describes the isolation and characterization of H-1 RF DNA as part of the continuing study of the mechanisms and control of DNA replication in the eukaryotic cell. The H-1 RF DNA is a linear duplex molecule containing the viral strand and its complement. The complementary strands of the RF DNA have been separated by equilibrium density gradient centrifugation. The RF DNA has a buoyant density of 1.705 in neutral CsCl and an estimated guanine plus cytosine (GC) content of 45.9%. It has a sedimentation coefficient of 17 S . The calculated molecular weight of 3.7 × 10 6 is twice that of the single-stranded virion DNA. H-1 virions contain DNA that is homogeneous and free of complementary strands.

Parvovirus H-1 expression: mapping of the abundant cytoplasmic transcripts and identification of promoter sites and overlapping transcription units

“…The supernatants were ethanol precipitated, suspended in 50 mM Tris hydrochloride-S mM EDTA (pH 7.9), and digested for 3 h at 37°C with 50 ,ug of RNase A (Sigma Chemical Co.) per ml which had previously been heat treated to inactivate any contaminating DNase. Three extractions with phenol-chloroform followed by ethanol precipitation resulted in a crude RF fraction consisting primarily of monomer-length H-1 RF DNA, with a smaller amount of H-1 multimer-length DNA (25). Monomer-length H-1 RF DNA was further purified by preparative-scale electrophoresis in 1% agarose gels.…”

“…Sequence of H-1 DNA in the vicinity of promoter regions. The sequence is reproduced from Rhode and Paradiso (25) to illustrate important restriction sites, as well as presumptive initiation and splice sites which are referred to in the text. (A) H-1 sequence in the vicinity of the left-end promoter (4 to 11 map units).…”

Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication

“…Thus, it is unlikely that inhibition of H-3 protein synthesis accounted for the inhibition of H-3 RF DNA synthesis caused by coinfection with ts14. The failure of ts14 to inhibit LuIII RF DNA synthesis was not due to LuIII inhibition of ts14 protein synthesis since LuIII RF DNA was not found to be inhibited for experiment 5 (c.f. experiment 2, Table 4) or for experiment 6 (comparative data not shown) of Table 5, where ts14 protein was predominant.…”

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

“…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.…”