John A. Holowczak

Replication of vaccinia DNA in mouse L cells I. In vivo DNA synthesis

Abstract Replication of vaccinia DNA was analyzed in lysates of intact, vaccinia-infected L cells prepared under conditions which preserved the structure of replicating and mature viral DNA molecules. The techniques employed permitted the separation of viral and host DNA, as confirmed by DNA-DNA hybridization. Sedimentation analysis in alkaline sucrose gradients showed that, during the period of maximum vaccinia DNA replication in L cells [2–3 hr postinfection (h.p.i.)], 10–12 S viral DNA fragments were preferentially labeled by short pulses (0.5–10 min) of [ 3 H]thymidine. About 20–30% of the pulse-labeled DNA was hydrolyzed by nuclease S,. These results support the view that viral DNA replication was discontinuous and may involve single-stranded DNA intermediates. Pulse-chase experiments showed that the 10–12 S fragments elongated into 30–50 S “intermediate-sized” DNA species and finally into 70–72 S (full length) viral DNA in about 30 min, which would require the incorporation of 6500 nucleotides/min. The conversion of mature viral DNA (70–72 S) into mature, cross-linked DNA (which sedimented at 92–94 S and 102–106 S in alkaline sucrose gradients) occurred late in infection (5–6 h.p.i.), when virion assembly had begun. Replicating viral DNA molecules were pulse-labeled with [ 3 H]thymidine and chased with bromodeoxyuridine (BrdU); the labeled DNA species were analyzed by equilibrium density centrifugation in CsCl. Hybrid (HL) molecules ( ϱ = 1.77 g/cm 3 ) were detected, demonstrating that viral DNA replication was semiconservative. Analysis of replicating viral DNA molecules in ethidium bromide-CsCl gradients at equilibrium failed to show the presence of circular or superhelical duplexes. This result and the fact that no viral DNA molecules of greater than unit length were labeled during long or short pulses suggest that viral DNA replication is symmetrical.

Structure of vaccinia DNA: Analysis of the viral genorne by restriction endonucleases

DNA from the WR strain of vaccinia virus was cleaved with five restriction endonucleases and the molecular weight of each restriction fragment was determined. From a summation of the molecular weights of these DNA fragments, the molecular weight of the vaccinia genome was estimated to be approximately 130 × 106. By electrophoresis in agarose gels under alkaline conditions, two HindIII fragments (WR-HindIII-34.8, WR-HindIII-22.7) and two SalI fragments (WR-SaI-3.5 and either a WR-SalI-1.1 fragment or WR-Sall-0.9) were identified as arising from the cross-linked terminal regions of the vaccinia virus genome. Heterogeneity in the size of these two fragments was observed upon cleavage of DNA purified from vaccinia virus (WR strain) which was serially passaged in various cell lines, but not in plaque-purified virus preparations. The structure of DNA from the CV-1 strain of vaccinia virus was also analyzed by cleavage with restriction endonucleases and compared to that of the WR strain; with the exception of the terminal fragments, all HindIII fragments observed in the WR digest were also observed in the CV-1 digest. Among the digestion products of vaccinia DNA, we have observed restriction fragments which are present in submolar quantities. The presence and size of these bands appear to depend upon the host cell in which the virus is propagated.

Uncoating of poxviruses: I. Detection and characterization of subviral particles in the uncoating process☆

Abstract Using highly purified, radioactively labeled virions, the uncoating of vaccinia virus, strain WR in HeLa S3 cells has been studied. When cytoplasm was prepared from infected cells 60–90 min after infection, in addition to particles which sedimented like complete virions (SubV-1), four fractions containing subviral particles could be resolved by velocity gradient centrifugation. These were labeled SubV-1A, SubV-2, SubV-3, and SubV-4. Uncoated DNA was recovered from the top of the gradient. The parental DNA in the subviral particles was resistant to DNase digestion, about 80% of the “uncoated” DNA was rendered acid soluble after treatment with DNase. Virions were degraded in vitro using a combination of detergent treatment and trypsin digestion techniques. Cores with lateral bodies and viral cores were isolated and partially purified. It could be shown that the major component of SubV-1A cosedimented with cores with lateral bodies and that in SubV-2 with viral cores. No in vitro degradation product could be prepared by the techniques described which cosedimented with the components of SubV-3 or SubV-4. Electron microscopic examination of particles from the SubV-4 fraction showed brick-shaped bodies similar to cores but which had lost part or all of their outer layers. These particles rather than rupturing under stress appeared to unravel suggesting the core membrane had been removed or altered. Using doubly labeled virions, it was estimated that about 12% of the core peptides were removed when the cores were converted to SubV-4 during uncoating. The kinetics of disappearance of each of the subviral particles and the appearance of “uncoated” DNA with time after infection was determined. Uncoating could be shown to proceed normally in the presence of cytosine arabinoside (100 μg/ml). When cells were treated with puromycin (100 μg/ml), protein synthesis was not completely inhibited as measured by the incorporation of 14C-amino acids into TCA-insoluble material. About 10–15% of the level measured in untreated cells persisted during the first 3–4 hours after infection and then slowly declined. Under these conditions uncoating was slowed, and only 35–40% of the parental viral DNA was uncoated. As protein synthesis became more completely arrested, the formation of SubV-4 was blocked and uncoating stopped. In the presence of Actidione which inhibited protein synthesis by 98% within 30 min after addition of the drug, uncoating did not occur. SubV-1A, SubV-2, and SubV-3 were formed but SubV-4 was never detected. The results show that the formation of SubV-4 and continued uncoating are cell mediated processes which depend on active protein synthesis.

Glycopeptides of vaccinia virus: I. Preliminary characterization and hexosamine content

Abstract When vaccinia virus, strain WR, was propagated in HeLa S3 cells in the presence of d -glucosamine-14C, the progeny virus, after purification, was found to be labeled. The specific activity of highly purified virus was 2.42 cpm/μg of viral protein. Such virus preparations were solubilized in SDS, urea and 2-mercaptoethanol and subjected to gel electrophoretic analysis. The major d -glucosamine-14C labeled moiety migrated in the gel with VSP-6, a peptide complex present on the surface of the virus, possibly in the outer membrane. Controlled degradation of labeled virus with Nonidet-P40 and 2-mercaptoethanol produced viral cores which after purification retained 6% of the radioactivity of the whole virus. Although label was associated with VSP-4 and VSP-5, the exact nature of the labeled peptide present in the core could not be determined. Acid hydrolysis of glucosamine-14C-labeled virions followed by column and paper chromatographic analysis showed that 90–94% of the d -glucosamine-14C label could be recovered as glucosamine. Ninhydrin degradation of the hexosamine fraction showed that 10% or less was in the form of galactosamine. Under the conditions employed no sialic acid residues were detected. Glucosamine could also be detected in hydrolyzates of cores prepared from glucosamine-14C-labeled virus. Because of the small amounts of radioactivity associated with purified cores, further characterization of the hexosamines present was not undertaken. When pronase digests of glucosamine-14C-labeled virus was analyzed by chromatography on Sephadex G-50 columns, two major fractions were resolved. These have not been fully characterized but may offer suitable material for carrying out detailed analysis of the carbohydrate portion of the glycoprotein. The incorporation of glucosamine-14C into TCA-insoluble material at various times after infection was determined. Incorporation closely paralleled total protein synthesis in infected cells suggesting that the glycopeptide is synthesized both early and late during the infection cycle.

Model for vaccinia virus DNA replication

Abstract Replicating vaccinia DNA molecules synthesized in vivo and in vitro, when examined in the electron microscope, were found to contain dsDNA loops which formed at one end of the molecules. A progressive increase in the size of the loop in individual molecules indicated that chain elongation was occurring. These results suggest that DNA replication was initiated at, and elongation proceeded from, one end of the viral DNA molecules. Based on these observations and previously published biochemical experiments, a model for vaccinia virus DNA replication is proposed.

Vaccinia virus transcription: hybridization of mRNA to restriction fragments of vaccinia DNA.

Abstract Differences in the transcription pattern of vaccinia mRNA synthesized in vivo and in vitro were revealed by hybridization of these RNAs to Hind III restriction fragments of vaccinia DNA that had been resolved by electrophoresis in agarose gels, denatured, and transferred to strips of nitrocellulose filters. Sequences homologous to all Hind III fragments were synthesized in vitro by Escherichia coli RNA polymerase in the presence of vaccinia DNA as a template. In vitro , the virion-associated RNA polymerase efficiently transcribed regions of the genome corresponding to all Hind III fragments except Hind III · J and Hind III · L. RNA synthesized in vivo in the presence of cycloheximide contained sequences homologous to all Hind III fragments including Hind lII · J but not Hind III · L. Sequences homologous to fragments J and L as well as all others were synthesized in vivo in the presence of hydroxyurea. It therefore appears that a region localized in Hind III · L is transcribed efficiently in vivo only after uncoating of the genome is completed. When RNAs synthesized at early (0–3 hr) or late (4–8 hr) times after infection in the absence of inhibitors were hybridized to Hind III fragments, some portion of nearly every fragment was found to be transcribed under both conditions. Densitometric analysis of autoradiograms of the filter strips revealed that the amount of RNA hybridizing to certain restriction fragments varied in the two samples. Through hybridization competition experiments in which labeled late RNAs were competed with unlabeled early RNAs, it was found that regions of the genome located within Hind lII fragments A, (D, E), and (G, H) are transcribed to a greater extent at late times than at early times after infection. Similarly, it was found that sequences hybridizing to Hind III fragments A, B, F, K, and M were more abundant in RNA synthesized by the core-associated RNA polymerase in vitro , than in “early” RNA synthesized in vivo .

Poxvirus DNA: I. Studies on the structure of the vaccinia genome

Abstract Vaccinia virions labeled with [3H]thymidine or [14C]thymidine were isolated and purified using two methods. In Method I, the virion preparations were sonicated during purification, banded repeatedly in sucrose gradients until their specific activity (cpm/μg viral DNA) was constant, and were frozen and thawed at least once before their DNA (DNA-I) was analyzed. In Method II virions were separated from other cellular constituents by a single banding in 20–40% linear sucrose gradients. The preparations were neither sonicated nor were they frozen before analysis of their DNA (DNA-II). DNA-I was released by solubilizing virions with detergents and 2-mercaptoethanol. In 5–20% neutral sucrose gradients DNA-I, released in this way, sedimented at about 72 S relative to Ad2 DNA. DNA-I denatured with alkali fragmented into three size classes of molecules, sedimenting at about 56–60, 45–51, and 27–32 S in alkaline sucrose gradients. Analysis of DNA-I purified by phenol extraction on hydroxyapatite or benzoylated napthoylated DEAE-cellulose (BND-cellulose) columns showed that while most of the sequences in DNA-I were base paired, DNA-I molecules also contained ssDNA or weakly hydrogen bonded regions which eluted from BND-cellulose with 2% caffeine and 0.1 N NaOH. DNA-II molecules released from virions by lysis with detergents and 2-mercaptoethanol sedimented at 68 S in neutral sucrose gradients relative to Ad2 DNA. After denaturation with alkali and analysis in alkaline sucrose gradients, DNA-II molecules sedimenting at 102–106 and 90–92 S, appropriate for closed ssDNA circular molecules and linear ssDNA molecules, respectively, with approximately twice the molecular weight expected for a single complimentary DNA strand from vaccinia genomes of MW 120–150 × 106 were detected. This result and other experimental data support the view that the DNA-II genome was cross-linked. Analysis of DNA-II preparations on hydroxyapatite and BND-cellulose columns demonstrated that 80% or more of the DNA-II genomes analyzed were completely double-stranded. Freezing and thawing or sonication of virions prepared by Method II resulted in alterations to the genomes so that they behaved upon sedimentation analysis like DNA-I genomes.

Biochemical and electron microscopic observations of vaccinia virus morphogenesis in HeLa cells after hydroxyurea reversal

Abstract Examination of thin sections of S3 HeLa cells propagated in suspension cultures and infected with vaccinia virus in the presence of hydroxyurea (HU) showed accumulation of immature virus particles. Upon removal of the drug the typical cytoplasmic development of virions was observed, confirming the usefulness of HU for separating early and late events in the replication cycle of poxvirions in spinner cultured cells and for studying viral morphogenesis under quasisynchronous conditions. Six hours after HU reversal, cytoplasm prepared by lysis of cells in Nonidet P-40 was fractionated by sucrose density centrifugation. Particles obtained by the fractionation procedure were negatively stained and their morphology characterized as follows: fraction I contained complete virions and cores with lateral bodies; fraction II contained particles 2300 × 3000 A in size which resembled viral cores but lacked some of the surface layers found on such subviral particles prepared in vitro; fraction III contained spherical particles 2600–2800 A in diameter and tubular structures of varying lengths and 1300 A in diameter; fraction IV yielded particles which morphologically resembled viral cores but had irregular surfaces and appeared damaged; their sedimentation behavior during density gradient centrifugation and subsequent isolation suggested that they may be membrane bound in the cytoplasm and were mechanically released during isolation. When cytoplasmic samples were centrifuged in velocity gradients under conditions where subviral and viral particles were pelleted, progeny DNA was found throughout the gradient. In sucrose gradients prepared in 10−3M phosphate buffer, pH 7.2, this DNA could be resolved into three classes. A minor fraction (10% or less of the radioactivity) remained at the top of the gradient, major components sedimented at 50–100 S (about 30–40%), the third > 100 S (10–15%). The remainder of the radioactivity was in the pellet. When the 50–100 S fraction was analyzed in alkaline gradients, the denatured DNA sedimented at about 30 S. Early after HU reversal (1–3 hr), before significant assembly of viral DNA into DNase resistant structures had occurred, 85–90% of the newly synthesized viral DNA could be recovered in the form of virosomes. Thin sections of viral DNA protein complexes when examined in the electron microscope were found to be heterogeneous in composition. Irregular ovoid bodies, 0.8–1.0 μm by 0.5 to 0.7 μm in size and filamentous material often in large aggregates composed of fibrils about 20 A in diameter comprised the major components observed in virosome preparations.

RNA synthesis in vaccinia-infected L cells: Inhibition of ribosome formation and maturation

Abstract The shutoff of cellular ribosome formation following infection by vaccinia virus was studied in L mouse fibroblasts. The synthesis of precursor rRNA, its methylation, and its conversion first to nuclear, and finally to cytoplasmic ribosomal particles was followed. The data show that by 2 hr after infection, the processing of precursor rRNA molecules and the rate of maturation of ribonucleoprotein particles was slowed. Three hours after infection little or no synthesis of new ribosomes was detected. The appearance of newly synthesized protein in cytoplasmic ribosomes was compared to that of new rRNA at various times after infection. The synthesis of new ribosomal protein was clearly depressed before that of new rRNA. Experiments in which cells were infected with amino acid- 14 C labeled virions showed that peptides derived from the infecting virions become associated with the nucleus and the nucleolus. It is postulated that the observed inhibition of cellular ribosomal synthesis may be mediated by such peptides.

The response of BHK21 cells to infection with type 12 adenovirus. V. Stimulation of cellular RNA synthesis and evidence for transcription of the viral genome.

Abstract The abortive infection of G1-arrested BHK21 cells with type 12 adenovirus (Ad12) has been shown to induce a 3- to 5-fold increase in the rate of uridine- 3 H incorporation into RNA, with a time course parallel to that of the induction of cellular DNA synthesis. Neither of these responses occurs in the absence of infectious virus. Uridine kinase activity is increased at the same time. Sucrose gradient analysis of the RNA has revealed that 45 S, 32-28 S, 18 S, and 4 S species of cellular RNA are all synthesized at an increased rate. Hybridization studies have demonstrated that Ad12-specific RNA is also synthesized. Its synthesis is first detectable at 10 hr and reaches a maximum at 12–14 hr after infection. It sediments in the 18 S region of the sucrose gradient and is presumably virus messenger.