Mariano Esteban

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.

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 .

Replication of vaccinia DNA in mouse L cells III. Intracellular forms of viral DNA

Abstract Parental and replicating vaccinia DNA molecules, labeled with [ 3 H]thymidine or [ 14 C]thymidine, present in infected L cells were analyzed by sedimentation in neutral and alkaline sucrose gradients. Alkaline sucrose gradient sedimentation analysis of labeled parental genomes present in infected cells showed that: (a) Such genomes were not degraded to acid-soluble products during the infection cycle; (b) cell-associated, cross-linked parental molecules (102 and 90–92 S) were “nicked”; parental DNA molecules sedimenting at 70–72 S were detected, but further nicking or degradation did not occur; and (c) molecules sedimenting at 90–92 S appeared to accumulate in the cytoplasm of infected cells and could serve as the templates for semiconservative DNA replication. When analyzed in neutral sucrose gradients, about 90% of viral DNA molecules labeled from 1 to 2 hr postinfection were associated with large aggregates or complexes which pelleted under the conditions of analysis used in these studies. With time (2–3 hr) after infection, viral DNA molecules could be dissociated from such aggregates and resolved by sedimentation in neutral sucrose gradients. The dissociation of labeled viral DNA molecules from complexes required continuous protein synthesis. Viral DNA could be released from complexes by treatment with alkali or digestion with S-1 nuclease, but not with RNase or Pronase. The results suggest that single-stranded (ss) DNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes.

Topography of vaccinia virus DNA

Abstract Contour length measurements in an electron microscope of vaccinia genomes released from virions by lysis in Sarkosyl and 2-mercaptoethanol at 4° on the surface of gradients, followed by sedimentation into the gradients to remove proteins, showed that the linear, double-stranded (ds) viral DNA molecules prepared in this way, had a MW of 132 ±5.93 × 10 6 . Digestion of the viral DNA with the endonuclease Eco RI, followed by electrophoretic analysis in 0.7% agarose gels, allowed 28 fragments to be resolved, ranging in MW from 14.0−0.4 × 10 6 . The sum of the MWs of these fragments was estimated to be 130–132 × 10 6 . Virions were lysed by brief treatment with Sarkosyl and 2-mercaptoethanol at 23° and the released viral DNA molecules, without further purification, were examined in an electron microscope. Hairpin-like structures observed along the length of the viral DNA disappeared after digestion with Pronase, showing that proteins held such structures in place. Loops, containing single-stranded (ss) DNA and dsDNA regions, and dsDNA branches observed in viral DNA molecules were not affected by Pronase digestion. These structures appear to be formed when the virion-associated ssDNA nuclease and (or) RNA polymerase, activated when virions are treated with detergent and 2-mercaptoethanol, function independently or in concert to destabilize selected regions in the DNA.

Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication.

Abstract The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [ 3 H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ϱ = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ϱ = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.

Replication of vaccinia DNA in mouse L cells. II. In vitro DNA synthesis in cytoplasmic extracts.

Abstract Cytoplasmic extracts prepared from vaccinia virus-infected L cells catalyzed the incorporation of labeled deoxynucleotide triphosphates into DNA which hybridized with vaccinia virus DNA. The incorporation of [ 3 H]thymidine 5′ triphosphate ([ 3 H]TTP) into DNA was shown to be dependent on the presence of all four deoxynucleoside triphosphates and incorporation was stimulated twofold by the addition of ATP, NAD, and ribonucleoside triphosphates. The incorporation of [ 3 H]TTP in vitro was linear for 10 min and continued at a reduced rate for 30 min at 30°. The viral DNA synthesized in vitro was analyzed by sedimentation in alkaline-sucrose gradients and by isopycnic centrifugation in CsCl gradients. Alkaline-sucrose sedimentation analysis showed that replication of in vitro labeled DNA was discontinuous. Small fragments (∼10 S) were synthesized in vitro in 10–30 sec which appeared to elongate so that after 30 min of synthesis the in vitro synthesized molecules cosedimented with in vivo labeled viral DNA species of 10–70 S. No molecules of greater length than mature viral single-stranded DNA (Type 1, 70–72 S) were observed when cell extracts prepared 3 hr postinfection were employed. Replication of viral DNA in vitro was symmetrical. No evidence for circular or superhelical DNA duplex molecules was obtained when in vitro synthesized DNA was analyzed by equilibrium density centrifugation in CsCl containing ethidium bromide.

Electron microscopic studies of transcriptional complexes released from vaccinia cores during RNA-synthesis in vitro: Methods for fractionation of transcriptional complexes

Electron microscopic (EM) and biochemical methods were employed to study the transcriptional complexes present in detergent lysates of vaccinia virus cores actively synthesizing RNA in vitro. When processed and examined in the EM, 14 transcriptional sites could be observed on full-length DNA templates. Fractionation of lysates by equilibrium density centrifugation in CsSO4, chromatography on hydroxyapatite columns or by sedimentation in sucrose gradients, allowed isolation of DNA templates associated with transcripts but these manipulations often resulted in fragmentation of the DNA template or promoted the release of transcripts from the template. It is suggested that RNA transcripts remain associated with the template in regions of supercoiling. These regions, in turn, may be maintained by DNA-protein interactions which are compromised as the transcriptional complexes are fractionated and purified.

Biochemical and electron microscopic studies of the transcription of vaccinia dna by rna polymerase from escherichia coli: Localization and characterization of transcriptional complexes

We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation. Electron microscopic studies of the interaction of E. coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with pre-early, early, and late in vivo RNA; self-annealing of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected. These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors. With extended in vitro incubations of the E. coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish. This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring. The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA. The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.