Viral nucleoprotein complexes in cells infected with the parvovirus MVM

Virus specific deoxyribonucleic acid (DNA) synthesis in BHK-21/C13 cells infected with the autonomous Parvovirus Minute Virus of Mice (MVM) can first be detected 8-10 hour post infection (hpi) and reaches a maximum rate of DM synthesis between 14-16 hpi. Viral DNA was extracted from infected cells, between 16-18 hpi, using a method which preserved protein and DNA interactions. The mechanisms of viral DNA replication and the assembly process were studied, under these conditions. A method was developed which quantitively and selectively extracted viral DNA from infected cells. This method relies on lysis of infected cells with the non-ionic detergent, NP40, followed by separation of the cells into cytoplasm and nuclei. The nuclei were then treated with various NaCl concentrations. Optimal release of viral DNA, without release of cellular DNA, occurred at a final concentration of 0.5M NaCl. The optimal pH for the extraction conditions was pH 7.0. Two nucleoprotein complexes were detected. A fast sedimenting, approximately lOOS, nucleoprotein complex, designated Cl, was present in the cytoplasmic extract. 01 had a buoyant density in CsCl slightly greater than that of mature virus and a sedimentation coefficient slightly less than mature virus. Mature MVM sediments at 110S. Cl is 20% sensitive to micrococcal nuclease digestion. The second nucleoprotein complex, designated Oil, sedimented at 22S with a leading edge towards higher sedimentation values. CII was located in the nucleus and constitutes the major viral DNA species in the infected cell. Protein was shown to be associated with the DNA by the change in sedimentation coefficient upon digestion with SDS and pronase, the increased sensitivity of the DNA to digestion with DNase I after treatment with agents which destroy protein and DNA interactions, and the association of 33-S-L-methionine labelled protein with the DNA. Reconstruction experiments have indicated that the association of protein with the DNA is not due to am artefact of the extraction procedure. The DNA components in the nucleoprotein complexes were analysed by neutral and alkaline agarose gel electrophoresis, Nal buoyant density analysis, sensitivity to digestion with SI nuclease and hybridisation analysis. Cl was shown to consist of single stranded DNA of the same molecular weight as MVM viral DNA. The DNA was demonstrated to be the viral strand and not the complementary strand by hybridisation with duplex MVM DNA synthesised vitro from MVM viral DNA using DNA polymerase I. The main band of CII consisted of a duplex DNA molecule twice the molecular weight of MVM viral DNA and therefore represents monomer replicative form (RP) DNA. When analysed under alkaline conditions the DNA contained molecules up to twice genome length. This indicated that some of the DNA in the duplex consisted of covalently linked viral and complementary strands. Confirmation of this was obtained by the observation that some of the GII DNA molecules were capable of spontaneous renaturation. The fast-sedimenting region of CII contained DNA twice and four times the length of monomer EP DNA, but, only contained DNA up to twice genome length under alkaline conditions. CII was shown to be viral in origin by displacement hybridisation. Pulse-chase experiments revealed that CII was a precursor to Cl although no replicative intermediate could be isolated. Preliminary electron microscopic studies revealed that the major DNA species was a linear double stranded DNA molecule of genome length. Potential replicative intermediates were identified by electron microscopy but no confirmatory data was obtained. In conclusion, viral DNA replication occurs in the form of nucleoprotein complexes and a putative maturation product was identified. The mechanisms of Parvovirus DNA replication and assembly are discussed