Biochemical and electron microscopic studies of the replication and composition of milker's node virus

Abstract

Replication of milker's node virus (MNV) DNA begins 4 to 8 h postinfection, continues to 30 to 36 h postinfection in the cytoplasm of infected, primary bovine embryonic kidney cells, and is accompanied by an inhibition of host nuclear DNA synthesis. Between 20 and 24 h postinfection, newly replicated genomes are incorporated into particles which cosediment with purified MNV. These biochemical measurements could be correlated with the development of MN virions as revealed by electron microscopic analysis of thin sections prepared from infected cells. Analysis of the DNA in purified MNV showed that the virions contained a double-stranded DNA molecule with a molecular weight of 85 x 10(6) to 87 x 10(6) and a guanine-plus-cytosine content of about 63%. After denaturation and sedimentation analysis of MNV DNA in alkaline sucrose gradients, three major DNA species were resolved. These species appeared to represent intact, terminally cross-linked genomes (approximately 75 to 80S); genomes bearing one nick (or with one cross-link removed) (60 to 65S); and complementary, denatured DNA strands released from cross-linked genomes bearing two nicks (or with both cross-links removed) (52 to 55S). Forty [35S]methionine-labeled polypeptides, ranging from approximately 200,000 daltons to 10,000 to 15,000 daltons, were detected by radioautography after polyacrylamide gel electrophoresis of the proteins present in detergent-solubilized MNV preparations. Treatment of MN virions with Nonidet P-40, beta-mercaptoethanol, and sonication released 10 polypeptides, which were apparently located on the surface of virions. Further fractionation of these released polypeptides, followed by electron microscopy and polyacrylamide gel electrophoresis, indicated that a 42,000- to 45,000-dalton polypeptide is a major component of the threadlike tubule structure present on the surface of MN virions.