Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis (original) (raw)

1977, Journal of Virology

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, tsl, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S. L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoradiography, and digestion with endo R EcoRI, was high. H-1 RF DNAs were linear doublestranded molecules, 1.53 ,m in length. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of OX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 x 106 to 1.59 x 106 (Berkowitz and Day, 1974). The parvovirus H-1 contains a singleceeds from the end containing the origin to the stranded (ss) DNA (22), and is capable of auton-opposite terminus at a uniform rate. The omous replication (12, 20, 21). During infection, branched RI DNA appeared to be entirely ds. a double-stranded (ds) replicative form (RF) Dimer-length RF DNA molecules and RIs were DNA is synthesized and replicated semiconser-also observed. The lengths of H-1 viral and RF vatively at a nearly exponential rate (13). Prog-DNA relative to those of pX174 were measured, eny ssDNA is produced simultaneously, pre-and the molecular weight of H-1 ssDNA was sumably by displacement from RF DNA engag-determined to be at least 1.48 x 106 to 1.59 x ing in asymmetric DNA synthesis (14), and 106, similar to the value obtained by gel electroencapsidated into virions. In this study we have phoresis (16). Additional data on the location of examined H-1 replicative form DNA and its the origin of replication has been obtained by replicative intermediates (RI DNA) with the partial denaturation mapping, and will be preelectron microscope to define the geometry of sented in the following paper of this series (19). RF DNA replication. To minimize any confusion with progeny viral DNA synthesis, we an-MATERIALS AND METHODS alyzed the replicating DNA of a temperature-Virus and cells. Parasynchronous cultures of secsensitive mutant of H-1, tsl, which is deficient ondary hamster embryo fibroblasts or human NB in progeny ssDNA synthesis, but not in RF cells were prepared and infected with tsl or wild-DNA replication (15). type (wt) H-1 as previously described (16). Esche-Previous studies using ethidium bromiderichia coli H-502 and H-4714, and OX174 (wt) and CsCl density gradient centrifugation, velocity am3 were kindly provided by R. L. Sinsheimer. sedimentation, and gel electrophoresis pro-Viral DNA preparation. H-1 virion DNA was exduced no evidence for covalently closed circular tracted from purified virus (12) labeled with H-1 RF DNA (13). Electron microscope visualiz-[3H]thymidine ([3H]TdR) by lysis in 0.2 N NaOH and ation of H-1 RF DNA reveals it to be a linear centrifugation in an alkaline sucrose gradient (16). ationlofuH-i R. DAm reveal. itatosbe a lin OX174 viral DNA was prepared as in (11), and molecule, 1.53 um in length. Analysis of RIs XX174 RF DNA was produced as outlined by Johnshows that they are Y-shaped, with the origin son and Sinsheimer (8). H-1 RF DNA was labeled of replication located no more than 0.15 genome and prepared as previously using the Hirt extraction lengths from one end, and that replication pro-with Pronase digestion (16). The Hirt supernatants