d. gen. ViroL (I979), 42, 223-229
223
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De Novo Synthesis of Two Classes o f D N A Induced by Vaccinia Virus Infection of HeLa Cells (Accepted 30 August I978) SUMMARY
Equilibrium density gradient centrifugation in CsCI confirms that DNA synthesized after vaccinia virus infection of HeLa cells is homogeneous in buoyant density and thus in base composition and is similar in this respect to bulk HeLa cell DNA. In contrast, rate sedimentation in alkaline sucrose gradients distinguishes two main classes of virus-induced DNA, neither of which can be equated with cell DNA synthesized in the same cultures prior to infection. The slower sedimenting class of virus-induced DNA co-sediments with DNA from purified virus particles: the second class sediments faster than pre-labelled cell DNA. Heterogeneity of virus-induced DNA does not result from fragmentation of radioactively labelled DNA, virus-mediated breakdown of cell DNA or association with either proteins or polyamines. Both slow and fast sedimenting classes of virusinduced DNA contain sequences complementary to all restriction endonuclease Hind III-specific fragments of the virus genome. The multiple species of DNA synthesized after infection are distinguished further by the effect of ethidium bromide. At a concentration which prevents the formation of infectious progeny virus, this compound inhibits selectively the de novo synthesis of that clasffof virusinduced DNA which sediments faster in alkaline sucrose gradients. The vaccinia virus genome has been shown to be a linear duplex DNA molecule for which mol. wt. estimates vary from 17o x IO6 (Sarov & Becker, I967) to 12o × IOn (Berns & Silverman, 197o). The G + C content of vaccinia virus DNA is sufficiently similar to that of mammalian cell DNA to make separation by isopycnic centrifugation impractical except after denaturation and selective reannealing (Jungwirth & Dawid, I967). Virus DNA has been shown to be present in cytoplasmic aggregates in vaccinia-infected cells (Cairns, 196o; Loh & Riggs, 1961; Harford et al. 1966) and several studies of virus DNA replication have been made using cytoplasmic fractions of such cells. Joklik & Becker (I964) observed that newly replicated virus DNA was associated with large aggregates which sedimented rapidly in neutral sucrose gradients although their stability was dependent on the presence of Mg ~+ ions. These aggregates have been shown to contain both replicated virus DNA and protein (Dahl & Kates, I97oa) and to be capable of virus-specific mRNA synthesis in vitro (Dahl & Kates, I97ob). Rapid sedimentation of such structures under these conditions appears to result from association of DNA with virus-specific proteins and treatment with pronase of SDS releases DNA having sedimentation characteristics similar to those of virion DNA in neutral sucrose gradients (Polisky & Kates, 1972). However, virus-induced and cell-specific DNA species can be separated easily by lysis of whole, infected cells and rate zonal centrifugation in alkaline sucrose gradients in which denatured DNA sediments in a random coil configuration at velocities proportional to the lengths of the molecules. Anomalous sedimentation behaviour in denaturing gradients and electron microscopic observations indicate that the complementary strands of the vaccinia virus 0022-1317/79/O000--2880 $02.00~) 1979 SGM
224
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genome are cross-linked covalently at or very near the termini (Geshelin & Berns, I974). The present report describes two main classes of vaccinia-induced DNA detected in denaturing gradients. Neither of these is related to cell DNA synthesized in the same cells prior to infection. The Lister strain of vaccinia was pock-purified by growth on the chorioaUantoic membrane of fertile hens eggs and the morphology of virus particles was checked by electron microscopy. Vaccinia virus was propagated, purified and used to infect HeLa cell monolayers essentially as described previously (Joklik, t962; Williamson & Archard, ~976). Stock cell cultures were propagated routinely in the absence of antibiotics. Media used for experimental cultures contained zoo #g/ml kanamycin, IOO #g/ml streptomycin and IOO units/ml penicillin. Infected or sham-infected HeLa cell monolayers were labelled in the DNA by incubation from 2 to 4 h p.i. in medium containing o.2 #Ci/ml 2-14C-thymidine (62 mCi/mmol) or I.O #Ci/ml 6-3H-thymidine (5ooo mCi/mmol) respectively. Most progeny vaccinia DNA is synthesized during this period (Joklik & Becker, 1964). The cells were washed and recovered by treatment with o.o2 % EDTA in phosphate buffered saline solution (PBS A), pooled and digested for 3 h at 37 °C with o.1% sodium n-lauroyl sarcosinate (Sarcosyl NL 35), o.1% pre-incubated pronase and o.o2 M-EDTA prepared in o'oi5 M-sodium chloride, o'oo15 M-sodium citrate, pH 7"3. The digests were dialysed overnight against similar buffer and diluted I/5o into 6o % (w/v) caesium chloride solution to give a final density of 1-76 g/ml. Aliquots of 4"5 ml were centrifuged at I8 °C and Izoooo g for 6o h and the resulting gradients fractionated by upward displacement. After the addition of I m g calf thymus DNA as carrier, fractions were examined for acid-precipitable radioactivity by liquid scintillation spectrometry essentially as described previously (Archard & Williamson, i97i ). Both 14C-labelled DNA from infected cells and 3H-labelled DNA from uninfected cells banded as single peaks. The peaks were almost coincident, having buoyant densities of about 1.7o g/ml (data not presented). These results are in agreement with the determinations of Jungwirth & Dawid (I967) and confirm that vaccinia virus-induced DNA is homogeneous in overall base composition and is similar in this respect to HeLa cell DNA. DNA was analysed in further experiments by rate sedimentation in 5 to 25 % (w/v) alkaline sucrose gradients. Gradients were prepared in polypropylene tubes (boiled previously for I h in o-ooI N-NaOH, o.I M-EDTA) by layering 2.2 ml vol. of sucrose solutions in o'7 M-NaC1, o'oo5 M-EDTA, o'3 N-NaOH (gradient vol. I I ml) and allowed to diffuse for 3 h at room temperature. HeLa cell monolayers, pre-labelled in the DNA by incubation for I6 h in medium containing o'25 #Ci/ml 6-3H-thymidine, were washed twice and re-incubated for 2 h in medium lacking the labelled precursor to reduce unincorporated radioactivity. The cultures were then infected, virus-induced DNA labelled by incubation from 2 to 4 h p.i. in medium containing 0"25 #Ci/ml 2-14C-thymidine and the cells recovered as before. Volumes of o'5 ml of I N-NaOH were floated on the gradients and 0"5 ml samples of cell suspension containing about 3 × I °5 cells and o.o2 M-EDTA were layered and the cells allowed to lyse in situ for 3o min. The gradients were centrifuged at i8 °C and I Ioooo g for 2 h, fractionated by upward displacement and the incorporated radioactivity determined as described (Fig. xa). DNA synthesized before infection and labelled with aH-thymidine was isolated as a single major peak. In contrast, DNA labelled in the same cells with 14C-thymidine during the period of maximum virus DNA synthesis was isolated as two, widely separated, major peaks. One peak sedimented faster and the other more slowly than the 8H-labelled, cell DNA. Additionally, there was evidence of minor peaks of 14C-labelled DNA sedimenting at intermediate velocities and possibly coincident with cell DNA. These results
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Pig. I. Rate sedimentation of DNA from vaccinia virus infected HeLa cells in alkaline sucrose gradients. Sedimentation is from left to right. (a) Cultures pre-labelled with ZH-thymidinefor 16 h (©--C), infected and labelled with 14C-thymidinefor 2 to 4 h p.i. (Q---O). The sedimentation of *H-thymidine-labelled DNA from purified vaccinia virions in similar gradients is shown also ( i - - i , 3H, d/rain x 1o-4). The arrow indicates the peak position of phage T4 DNA. (b) Cultures labelled with a~C-thymidine from 2 to 4 h p.i. Ethidium bromide was present from I h p.i. at a concentration of io/*g/ml (O---O), 2o/zg/ml ( i - - A ) , 30/*g/ml (O--O), or 50/zg/ml (A--A). show that D N A synthesized in infected cells during the period of maximum virus D N A synthesis and homogeneous on the basis of equilibrium buoyant density may be resolved as two major fractions by rate sedimentation. Neither of these is coincident with H e L a cell D N A labelled before infection. Apparent, multiple species of D N A detected after infection do not result from either degradation or re-utilization of pre-labelled, cell D N A (Parkhurst et al. I973) as only the labelled thymidine supplied and incorporated during the period of virus D N A synthesis is found at these positions in the gradients. Additionally, such heterogeneity does not result from spontaneous fragmentation of all-labelled D N A (Peterson & Fox, I97I ) as similar sedimentation profiles are obtained when the labelling is reversed. In additional experiments, vaccinia-infected H e L a cell monolayers were labelled from 2 to 4 h p.i. with ZH-thymidine (2-o/,Ci/ml) plus either 14C-leucine (62 m C i / m m o l , o'5 #Ci/ml) or *4C-spermidine 0 2 2 m C i / m m o l , o'5/zCi/ml) and analysed in denaturing sucrose gradients as before. Radioactivity supplied as either 14C-leucine or 14C-spermidine did not co-sediment with incorporated aH-thymidine but remained near the top of the gradients (data not presented). These results indicate that the sedimentation characteristics of virus-induced
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Fig. 2. Hybridization of 3zP-labelled DNA to restriction endonuclease Hind III-specific fragments of vaccinia virus DNA (series A) or of HeLa cell DNA (series B) transferred to nitrocellulose membrane filter after separation by electrophoresis in agarose gels. Probes were prepared by nick translation of DNA isolated from the slow (1) or fast sedimenting (2) peak of DNA induced by vacciniaSrrfection of HeLa cells, from purified vaccinia virus particles (3) or from non-infected HeLa cells (4). D N A species from vaccinia-infected cells do not result from association with either proteins or polyamines. Vaccinia virus grown in the presence of 3H-thymidine, purified by isopycnic centrifugation in potassium tartrate density gradients and analysed under similar conditions yielded D N A co-sedimenting with the slower sedimenting D N A induced in H e L a cells by virus infection. The peak position of T 4 bacteriophage D N A labelled with either aH-thymidine or z~P-orthophosphate is indicated by the superimposed arrow. Denatured D N A from vaccinia virus particles sediments often but not invariably in alkaline gradients as a bifurcated peak. I f the mol. wt. of T4 D N A is taken as I3O × IOn with a sedimentation coefficient of about 7oS in denaturing gradients (Studier, 1965) then themajor component of D N A f r o m isolated vaccinia virus particles sediments at 98S representing a single strand of 15o × IO° mol. wt. The minor component presumably represents a single stranded circle of the same molecular weight as the cross-linked, native genome from which the 98S form is generated in denaturing conditions by a single nick (Geshelin & Berns, 1974). The specificity of D N A sedimenting at various positions in denaturing gradients was determined by hybridization to restriction endonuclease Hind III-specific fragments of vaccinia virus genome D N A , prepared essentially as described by Mtiller et al. (I 978), and of
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HeLa cell DNA. Restriction fragments were separated by electrophoresis in 0.6 ~o agarose slab gels, denatured and transferred to nitrocellulose membrane filters (Southern, 1975). Fractions containing the slow and fast sedimenting peaks of virus-induced DNA from infected cells were taken from gradients similar to that described in Fig. I (a) and the DNA recovered by ethanol precipitation after neutralization with HC1 in the presence of IOO mr~-tris-HC1, pH 7"4. DNA was labelled by nick translation essentially as described by Rigby et al. (1977). In addition to the recovered DNA, reaction mixtures (IOO/A) contained 20 /zM-deoxyribonucleotide triphosphates including 2. 5 #Ci each of (a32p)-d ATP and (c~z2P)-d CTP (specific activities I5o Ci/mmol), 5o mM-tris-HC1, pH 7"4, 5 mM-MgC12, 1 mM-fl-mercaptoethanol, 5 #g BSA and 2 units of DNA polymerase I (Boehringer Corp.); DNase was omitted. Nick translation was at 14 °C for 2 h and DNA was re-isolated by exclusion chromatography on Sephadex G5o followed by ethanol precipitation. Labelled DNA was denatured at lO5 °C for 5 rain and 200o0 to 5o0o0 Cerenkov ct/min were hybridized for 6o h to representative nitrocellulose strips bearing transferred, restricted vaccinia virus or HeLa cell DNA. Pre-treatment of strips, conditions of hybridization and subsequent washing were similar to those described by Jeffreys & Havell (1977). Strips were autoradiographed for 3 to IO days at - 7 o °C using pre-sensitized Fuji Rx X-ray film and an Ilford fast tungstate intensifying screen. DNA recovered from gradient fractions containing either the slow or fast sedimenting peak of virus-induced DNA from infected cells hybridized specifically to all Hind III restriction fragments of known vaccinia virus DNA to which control, nick translated, vaccinia virus total DNA hybridized also (Fig. 2a). Control 32p_ labelled HeLa cell DNA failed to hybridize to vaccinia DNA under these conditions. These results indicate that DNA synthesized during the period of maximum virus DNA replication in vaccinia-infected cells and either co-sedimenting with the vaccinia genome or sedimenting faster than #t vivo pre-labelled cell DNA contains sequences complementary to all Hind III restriction fragments of the virus genome. In contrast, such DNA failed to hybridize strongly to Hind III restricted HeLa cell DNA demonstrating that both the slow and fast sedimenting peaks of DNA induced by virus infection consist mainly of virus-specific sequences (Fig. 2b). In further experiments, infected HeLa cell monolayers were maintained from 1 h p.i. in media containing various concentrations of ethidium bromide. From 2 to 4 h p.i., the media also contained o'25 #Ci/m114C-thymidine. The cells were recovered and washed and samples analysed in alkaline sucrose gradients as before (Fig. I b). In the presence of IO/zg/ml ethidium bromide, the sedimentation profile of virus-induced DNA was similar to that found with non-inhibited, infected cells. In the presence of 2o/~g/ml or 30/zg/ml ethidium bromide, the amount of incorporated radioactivity present in the faster sedimenting peak was inhibited markedly while that in the slower sedimenting peak was relatively unaffected. At an inhibitor concentration of 50 #g/ml, the incorporation of radioactivity into the faster sedimenting peak was inhibited almost completely. These results confirm that two classes of DNA, other than cell DNA, are synthesized after infection and indicate that the synthesis of one such class is particularly sensitive to inhibition by ethidium bromide. This intercalating agent is preferentially bound by circular DNA (Radloff et al. 1967) and has been shown previously to inhibit the growth of vaccinia virus (Vilaginbs, 197o) or the replication of DNA in cytoplasmic fractions of vaccinia-infected cells (Raya & Vilaginbs, I97O). In the present study, the presence of 2o #g/ml ethidium bromide from 1 h p.i. completely inhibited the production of infectious progeny virus in vaccinia-infected HeLa cells (data not presented) suggesting that the fast sedimenting form of virus-induced DNA observed in denaturing gradients is essential to virus replication.
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As no evidence was obtained for association o f protein with the fast sedimenting material it seems unlikely that this is equivalent to the large aggregates observed previously in neutral gradients (Dahl & Kates, ~97oa) or that pronase treatment could result in conversion to a f o r m co-sedimenting with the virus genome (Polisky & Rates, ~972). Hybridization data suggest strongly that fast sedimenting D N A induced by vaccinia infection consists mainly o f virus-specific sequences. The presence o f some cell-specific sequences cannot be excluded although the material failed to hybridize strongly to H e L a cell D N A . H o w ever, degradation o f cell-specific, nascent D N A by a virion nuclease leading to inhibition of host nuclear D N A synthesis after vaccinia infection (Pogo & Dales, 1974)makes it unlikely that sedimentation characteristics result from association o f virus D N A with very high mol. wt. cell D N A . The separation described depends on the size and conformation o f D N A molecules rather than their density. The slower sedimenting peak from infected cells appears to correspond to the virus genome and the faster-sedimenting peak to a class o f virus-induced D N A which is larger or whose conformation, resistant to alkaline denaturation, favours rapid sedimentation and preferential binding o f ethidium bromide. The sedimentation behaviour o f vaccinia virus genome D N A in denaturing conditions is anomalous as the complementary strands o f the duplex are linked covalently in a m a n n e r reminiscent o f a continuous single strand polynucleotide chain. N i c k translation of fast sedimenting material f r o m denaturing gradients yields labelled D N A sequences representative o f the total virus genome sugesting that, in this case also, strand separation is not complete and t h a t rapid renaturation occurs after neutralization. It is suggested that the fast sedimenting material described here m a y be a partially circular replication complex o f this unusual virus genome.
Department of Virology St Mary's Hospital Medical School Paddington, London W2 IPG
L . C . ARCH~atD
REFERENCES ARCheD, L. C. & WILLt~MSON,J. D. 097I). The effect of arginine deprivation on the replication of vaccinia virus. Journal of General Virology I2, 249-258.
~E~',~S,K. r. & SlLVEaMAr~,¢. 097O). Natural occurrence of cross-linked vaccinia virus deoxyribonucleic acid. Journal of Virology 5, 299-3o4. CAIRNS, J. (I960). The initiation of vaccinia infection. Virology ix, 6o3-634. Dm-n., a. & RATES,J. R. (I970a). Intracellular structures containing vaccinia D N A : isolation and characterization. Virology 42, 453-462.
DANE,R. & ~TES, J. R. 097ob). Synthesis of vaccinia virus 'early' and 'late' messengel RNA in vitro with nucleoprotein structures isolated from infected cells. Virology 42, 463-472. OESn~HN,P. & BERNS,K. I. (I974). Characterization and localisation of the naturally occurring cross-links in vaccinia virus DNA. Journal of Molecular Biology 88, 785-796. HARFORD,C. G., HAMLIN,A. & RmDEa.S,E. 0966). Electron microscopic autoradiography of DNA synthesis in cells infected with vaccinia virus. Experimental Cell Research 42, 5o-57. JEFFREYS,A. S. & FLAWLL,R. A. (I977). A physical map of the DNA regions flanking the rabbit fl-globin gene. Cell x~, 429-439. JOICLK,W. K. 0962). The purification of four strains of poxvirus. Virology IS, 9-I8. JOKHK, W.K. & BECI~ER,Y. (I964). The replication and coating of vaccinia DNA. Journal of Molecular IBiology xo, 452-474. JUN~WIRTrI,C. & OAWIO,I. 13.(1967). Vaccinia DNA: separation of virus from host cell DNA. Archiv fi~r die gesamte Virusforschung ~-o, 464-468. Lon, P. c. & ~ G s , s. L. (I96I). Demonstration of the sequential development of vaccinial antigens and virus in infected cells: observations with cytochemical and differential fluorescent proceedures. Journal of Experimental Medicine Ix4, I49-I6O. Mf0LLER, H. K., WITTEK, R., SCHAFFNER.,W., SCHIJMPERL1,D., MENNA, A. & WYLER, R. (I978). Comparison of five poxvirus genomes by analysis with restriction endonucleases Hind Ill, Bam I and EcoRI. Journal of General Virology 38, 135-I47.
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PARKH--tJRST, J. R., PETERSON, A. R. & HEIDELBERGER, C. (1973)- Breakdown of HeLa cell D N A mediated by
vaccinia virus. Proceedings of the National Academy of Sciences of the United States of America 7o, 3200-3204, PETERSON, A. R. & FOX, B. W. (1971). Toxicity of tritiated thymidine in P388F lymphoma cells. II. Effects on D N A molecular weight. International Journal of Radiation Biology 19, 237-246. POGO, B. G. T. & DALES,S. (1974). Biogenesis of poxviruses : further evidence for inhibition of host and virus DNA synthesis by a component of the invading inoculum particle. Virology 58, 377-386. POLISKY, B. & KATES, J. (I972). Vaccinia virus intracellular DNA-protein complex: biochemical characteristics of associated protein. Virology 49, I68-I79. RADLOFF, R., BAUER,W. & VINOGRAD,J. (1967). A dye-buoyant-density method for the selection and isolation of closed circular duplex D N A : The closed circular D N A in HeLa cells. Proceedings of the National Academy of Sciences of the United States of America 57, I514-152I. RAYA, J. M. & VILAGINlbS,a. (I970). Action du bromhydrate d'6thidium sur le d6veloppement du virus vaccinal dans les cellules en culture de rein de babouin. II. Action sur les synth6ses d ' A D N et d ' A R N de cellules infect6es et non infect6es. Archly fi~r die gesamte Virusforschung 3o, 155-I 58. RIGBY, P. W. J., DIECKMANN,M., RHODES, C. & BERG, P. (I977). Labeling deoxyribonucleic acid to high specific
activity in vitro by nick translation with DNA polymerase I. Journal of Alolecular Biology xx3, 237-25I. SAROV,I. & EECI(ER, Y. (I967). Studies on vaccinia virus DNA. Virology 33, 369-375. SoLrrHERN, E. M. 0975). Detection of specific sequences among D N A fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 5o3-518. STUDIER, V. W. 0965). Sedimentation studies of the size and shape of DNA. Journal of Molecular Biology xI, 373-39o. VILAGIN~S, R. (I970). Action du bromohydrate d'6thidium sur le d6veloppement du virus vaccinial dans les cellules en culture de rein de babouin. I. Action sur la cin6tique de d6veloppement viral. Archivf~r die gesamte Virusforschurg 3o, 59-66. WILLIAMSON,J. O. & ARCH_'.RD,L. C. (1976). The effect of canaline on some events in vaccinia virus replication. Journal of General Virology 3o, 81--89.
(Received I7 M a y I977)
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De Novo Synthesis of Two Classes o f D N A Induced by Vaccinia Virus Infection of HeLa Cells (Accepted 30 August I978) SUMMARY
Equilibrium density gradient centrifugation in CsCI confirms that DNA synthesized after vaccinia virus infection of HeLa cells is homogeneous in buoyant density and thus in base composition and is similar in this respect to bulk HeLa cell DNA. In contrast, rate sedimentation in alkaline sucrose gradients distinguishes two main classes of virus-induced DNA, neither of which can be equated with cell DNA synthesized in the same cultures prior to infection. The slower sedimenting class of virus-induced DNA co-sediments with DNA from purified virus particles: the second class sediments faster than pre-labelled cell DNA. Heterogeneity of virus-induced DNA does not result from fragmentation of radioactively labelled DNA, virus-mediated breakdown of cell DNA or association with either proteins or polyamines. Both slow and fast sedimenting classes of virusinduced DNA contain sequences complementary to all restriction endonuclease Hind III-specific fragments of the virus genome. The multiple species of DNA synthesized after infection are distinguished further by the effect of ethidium bromide. At a concentration which prevents the formation of infectious progeny virus, this compound inhibits selectively the de novo synthesis of that clasffof virusinduced DNA which sediments faster in alkaline sucrose gradients. The vaccinia virus genome has been shown to be a linear duplex DNA molecule for which mol. wt. estimates vary from 17o x IO6 (Sarov & Becker, I967) to 12o × IOn (Berns & Silverman, 197o). The G + C content of vaccinia virus DNA is sufficiently similar to that of mammalian cell DNA to make separation by isopycnic centrifugation impractical except after denaturation and selective reannealing (Jungwirth & Dawid, I967). Virus DNA has been shown to be present in cytoplasmic aggregates in vaccinia-infected cells (Cairns, 196o; Loh & Riggs, 1961; Harford et al. 1966) and several studies of virus DNA replication have been made using cytoplasmic fractions of such cells. Joklik & Becker (I964) observed that newly replicated virus DNA was associated with large aggregates which sedimented rapidly in neutral sucrose gradients although their stability was dependent on the presence of Mg ~+ ions. These aggregates have been shown to contain both replicated virus DNA and protein (Dahl & Kates, I97oa) and to be capable of virus-specific mRNA synthesis in vitro (Dahl & Kates, I97ob). Rapid sedimentation of such structures under these conditions appears to result from association of DNA with virus-specific proteins and treatment with pronase of SDS releases DNA having sedimentation characteristics similar to those of virion DNA in neutral sucrose gradients (Polisky & Kates, 1972). However, virus-induced and cell-specific DNA species can be separated easily by lysis of whole, infected cells and rate zonal centrifugation in alkaline sucrose gradients in which denatured DNA sediments in a random coil configuration at velocities proportional to the lengths of the molecules. Anomalous sedimentation behaviour in denaturing gradients and electron microscopic observations indicate that the complementary strands of the vaccinia virus 0022-1317/79/O000--2880 $02.00~) 1979 SGM
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genome are cross-linked covalently at or very near the termini (Geshelin & Berns, I974). The present report describes two main classes of vaccinia-induced DNA detected in denaturing gradients. Neither of these is related to cell DNA synthesized in the same cells prior to infection. The Lister strain of vaccinia was pock-purified by growth on the chorioaUantoic membrane of fertile hens eggs and the morphology of virus particles was checked by electron microscopy. Vaccinia virus was propagated, purified and used to infect HeLa cell monolayers essentially as described previously (Joklik, t962; Williamson & Archard, ~976). Stock cell cultures were propagated routinely in the absence of antibiotics. Media used for experimental cultures contained zoo #g/ml kanamycin, IOO #g/ml streptomycin and IOO units/ml penicillin. Infected or sham-infected HeLa cell monolayers were labelled in the DNA by incubation from 2 to 4 h p.i. in medium containing o.2 #Ci/ml 2-14C-thymidine (62 mCi/mmol) or I.O #Ci/ml 6-3H-thymidine (5ooo mCi/mmol) respectively. Most progeny vaccinia DNA is synthesized during this period (Joklik & Becker, 1964). The cells were washed and recovered by treatment with o.o2 % EDTA in phosphate buffered saline solution (PBS A), pooled and digested for 3 h at 37 °C with o.1% sodium n-lauroyl sarcosinate (Sarcosyl NL 35), o.1% pre-incubated pronase and o.o2 M-EDTA prepared in o'oi5 M-sodium chloride, o'oo15 M-sodium citrate, pH 7"3. The digests were dialysed overnight against similar buffer and diluted I/5o into 6o % (w/v) caesium chloride solution to give a final density of 1-76 g/ml. Aliquots of 4"5 ml were centrifuged at I8 °C and Izoooo g for 6o h and the resulting gradients fractionated by upward displacement. After the addition of I m g calf thymus DNA as carrier, fractions were examined for acid-precipitable radioactivity by liquid scintillation spectrometry essentially as described previously (Archard & Williamson, i97i ). Both 14C-labelled DNA from infected cells and 3H-labelled DNA from uninfected cells banded as single peaks. The peaks were almost coincident, having buoyant densities of about 1.7o g/ml (data not presented). These results are in agreement with the determinations of Jungwirth & Dawid (I967) and confirm that vaccinia virus-induced DNA is homogeneous in overall base composition and is similar in this respect to HeLa cell DNA. DNA was analysed in further experiments by rate sedimentation in 5 to 25 % (w/v) alkaline sucrose gradients. Gradients were prepared in polypropylene tubes (boiled previously for I h in o-ooI N-NaOH, o.I M-EDTA) by layering 2.2 ml vol. of sucrose solutions in o'7 M-NaC1, o'oo5 M-EDTA, o'3 N-NaOH (gradient vol. I I ml) and allowed to diffuse for 3 h at room temperature. HeLa cell monolayers, pre-labelled in the DNA by incubation for I6 h in medium containing o'25 #Ci/ml 6-3H-thymidine, were washed twice and re-incubated for 2 h in medium lacking the labelled precursor to reduce unincorporated radioactivity. The cultures were then infected, virus-induced DNA labelled by incubation from 2 to 4 h p.i. in medium containing 0"25 #Ci/ml 2-14C-thymidine and the cells recovered as before. Volumes of o'5 ml of I N-NaOH were floated on the gradients and 0"5 ml samples of cell suspension containing about 3 × I °5 cells and o.o2 M-EDTA were layered and the cells allowed to lyse in situ for 3o min. The gradients were centrifuged at i8 °C and I Ioooo g for 2 h, fractionated by upward displacement and the incorporated radioactivity determined as described (Fig. xa). DNA synthesized before infection and labelled with aH-thymidine was isolated as a single major peak. In contrast, DNA labelled in the same cells with 14C-thymidine during the period of maximum virus DNA synthesis was isolated as two, widely separated, major peaks. One peak sedimented faster and the other more slowly than the 8H-labelled, cell DNA. Additionally, there was evidence of minor peaks of 14C-labelled DNA sedimenting at intermediate velocities and possibly coincident with cell DNA. These results
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Pig. I. Rate sedimentation of DNA from vaccinia virus infected HeLa cells in alkaline sucrose gradients. Sedimentation is from left to right. (a) Cultures pre-labelled with ZH-thymidinefor 16 h (©--C), infected and labelled with 14C-thymidinefor 2 to 4 h p.i. (Q---O). The sedimentation of *H-thymidine-labelled DNA from purified vaccinia virions in similar gradients is shown also ( i - - i , 3H, d/rain x 1o-4). The arrow indicates the peak position of phage T4 DNA. (b) Cultures labelled with a~C-thymidine from 2 to 4 h p.i. Ethidium bromide was present from I h p.i. at a concentration of io/*g/ml (O---O), 2o/zg/ml ( i - - A ) , 30/*g/ml (O--O), or 50/zg/ml (A--A). show that D N A synthesized in infected cells during the period of maximum virus D N A synthesis and homogeneous on the basis of equilibrium buoyant density may be resolved as two major fractions by rate sedimentation. Neither of these is coincident with H e L a cell D N A labelled before infection. Apparent, multiple species of D N A detected after infection do not result from either degradation or re-utilization of pre-labelled, cell D N A (Parkhurst et al. I973) as only the labelled thymidine supplied and incorporated during the period of virus D N A synthesis is found at these positions in the gradients. Additionally, such heterogeneity does not result from spontaneous fragmentation of all-labelled D N A (Peterson & Fox, I97I ) as similar sedimentation profiles are obtained when the labelling is reversed. In additional experiments, vaccinia-infected H e L a cell monolayers were labelled from 2 to 4 h p.i. with ZH-thymidine (2-o/,Ci/ml) plus either 14C-leucine (62 m C i / m m o l , o'5 #Ci/ml) or *4C-spermidine 0 2 2 m C i / m m o l , o'5/zCi/ml) and analysed in denaturing sucrose gradients as before. Radioactivity supplied as either 14C-leucine or 14C-spermidine did not co-sediment with incorporated aH-thymidine but remained near the top of the gradients (data not presented). These results indicate that the sedimentation characteristics of virus-induced
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Fig. 2. Hybridization of 3zP-labelled DNA to restriction endonuclease Hind III-specific fragments of vaccinia virus DNA (series A) or of HeLa cell DNA (series B) transferred to nitrocellulose membrane filter after separation by electrophoresis in agarose gels. Probes were prepared by nick translation of DNA isolated from the slow (1) or fast sedimenting (2) peak of DNA induced by vacciniaSrrfection of HeLa cells, from purified vaccinia virus particles (3) or from non-infected HeLa cells (4). D N A species from vaccinia-infected cells do not result from association with either proteins or polyamines. Vaccinia virus grown in the presence of 3H-thymidine, purified by isopycnic centrifugation in potassium tartrate density gradients and analysed under similar conditions yielded D N A co-sedimenting with the slower sedimenting D N A induced in H e L a cells by virus infection. The peak position of T 4 bacteriophage D N A labelled with either aH-thymidine or z~P-orthophosphate is indicated by the superimposed arrow. Denatured D N A from vaccinia virus particles sediments often but not invariably in alkaline gradients as a bifurcated peak. I f the mol. wt. of T4 D N A is taken as I3O × IOn with a sedimentation coefficient of about 7oS in denaturing gradients (Studier, 1965) then themajor component of D N A f r o m isolated vaccinia virus particles sediments at 98S representing a single strand of 15o × IO° mol. wt. The minor component presumably represents a single stranded circle of the same molecular weight as the cross-linked, native genome from which the 98S form is generated in denaturing conditions by a single nick (Geshelin & Berns, 1974). The specificity of D N A sedimenting at various positions in denaturing gradients was determined by hybridization to restriction endonuclease Hind III-specific fragments of vaccinia virus genome D N A , prepared essentially as described by Mtiller et al. (I 978), and of
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HeLa cell DNA. Restriction fragments were separated by electrophoresis in 0.6 ~o agarose slab gels, denatured and transferred to nitrocellulose membrane filters (Southern, 1975). Fractions containing the slow and fast sedimenting peaks of virus-induced DNA from infected cells were taken from gradients similar to that described in Fig. I (a) and the DNA recovered by ethanol precipitation after neutralization with HC1 in the presence of IOO mr~-tris-HC1, pH 7"4. DNA was labelled by nick translation essentially as described by Rigby et al. (1977). In addition to the recovered DNA, reaction mixtures (IOO/A) contained 20 /zM-deoxyribonucleotide triphosphates including 2. 5 #Ci each of (a32p)-d ATP and (c~z2P)-d CTP (specific activities I5o Ci/mmol), 5o mM-tris-HC1, pH 7"4, 5 mM-MgC12, 1 mM-fl-mercaptoethanol, 5 #g BSA and 2 units of DNA polymerase I (Boehringer Corp.); DNase was omitted. Nick translation was at 14 °C for 2 h and DNA was re-isolated by exclusion chromatography on Sephadex G5o followed by ethanol precipitation. Labelled DNA was denatured at lO5 °C for 5 rain and 200o0 to 5o0o0 Cerenkov ct/min were hybridized for 6o h to representative nitrocellulose strips bearing transferred, restricted vaccinia virus or HeLa cell DNA. Pre-treatment of strips, conditions of hybridization and subsequent washing were similar to those described by Jeffreys & Havell (1977). Strips were autoradiographed for 3 to IO days at - 7 o °C using pre-sensitized Fuji Rx X-ray film and an Ilford fast tungstate intensifying screen. DNA recovered from gradient fractions containing either the slow or fast sedimenting peak of virus-induced DNA from infected cells hybridized specifically to all Hind III restriction fragments of known vaccinia virus DNA to which control, nick translated, vaccinia virus total DNA hybridized also (Fig. 2a). Control 32p_ labelled HeLa cell DNA failed to hybridize to vaccinia DNA under these conditions. These results indicate that DNA synthesized during the period of maximum virus DNA replication in vaccinia-infected cells and either co-sedimenting with the vaccinia genome or sedimenting faster than #t vivo pre-labelled cell DNA contains sequences complementary to all Hind III restriction fragments of the virus genome. In contrast, such DNA failed to hybridize strongly to Hind III restricted HeLa cell DNA demonstrating that both the slow and fast sedimenting peaks of DNA induced by virus infection consist mainly of virus-specific sequences (Fig. 2b). In further experiments, infected HeLa cell monolayers were maintained from 1 h p.i. in media containing various concentrations of ethidium bromide. From 2 to 4 h p.i., the media also contained o'25 #Ci/m114C-thymidine. The cells were recovered and washed and samples analysed in alkaline sucrose gradients as before (Fig. I b). In the presence of IO/zg/ml ethidium bromide, the sedimentation profile of virus-induced DNA was similar to that found with non-inhibited, infected cells. In the presence of 2o/~g/ml or 30/zg/ml ethidium bromide, the amount of incorporated radioactivity present in the faster sedimenting peak was inhibited markedly while that in the slower sedimenting peak was relatively unaffected. At an inhibitor concentration of 50 #g/ml, the incorporation of radioactivity into the faster sedimenting peak was inhibited almost completely. These results confirm that two classes of DNA, other than cell DNA, are synthesized after infection and indicate that the synthesis of one such class is particularly sensitive to inhibition by ethidium bromide. This intercalating agent is preferentially bound by circular DNA (Radloff et al. 1967) and has been shown previously to inhibit the growth of vaccinia virus (Vilaginbs, 197o) or the replication of DNA in cytoplasmic fractions of vaccinia-infected cells (Raya & Vilaginbs, I97O). In the present study, the presence of 2o #g/ml ethidium bromide from 1 h p.i. completely inhibited the production of infectious progeny virus in vaccinia-infected HeLa cells (data not presented) suggesting that the fast sedimenting form of virus-induced DNA observed in denaturing gradients is essential to virus replication.
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As no evidence was obtained for association o f protein with the fast sedimenting material it seems unlikely that this is equivalent to the large aggregates observed previously in neutral gradients (Dahl & Kates, ~97oa) or that pronase treatment could result in conversion to a f o r m co-sedimenting with the virus genome (Polisky & Rates, ~972). Hybridization data suggest strongly that fast sedimenting D N A induced by vaccinia infection consists mainly o f virus-specific sequences. The presence o f some cell-specific sequences cannot be excluded although the material failed to hybridize strongly to H e L a cell D N A . H o w ever, degradation o f cell-specific, nascent D N A by a virion nuclease leading to inhibition of host nuclear D N A synthesis after vaccinia infection (Pogo & Dales, 1974)makes it unlikely that sedimentation characteristics result from association o f virus D N A with very high mol. wt. cell D N A . The separation described depends on the size and conformation o f D N A molecules rather than their density. The slower sedimenting peak from infected cells appears to correspond to the virus genome and the faster-sedimenting peak to a class o f virus-induced D N A which is larger or whose conformation, resistant to alkaline denaturation, favours rapid sedimentation and preferential binding o f ethidium bromide. The sedimentation behaviour o f vaccinia virus genome D N A in denaturing conditions is anomalous as the complementary strands o f the duplex are linked covalently in a m a n n e r reminiscent o f a continuous single strand polynucleotide chain. N i c k translation of fast sedimenting material f r o m denaturing gradients yields labelled D N A sequences representative o f the total virus genome sugesting that, in this case also, strand separation is not complete and t h a t rapid renaturation occurs after neutralization. It is suggested that the fast sedimenting material described here m a y be a partially circular replication complex o f this unusual virus genome.
Department of Virology St Mary's Hospital Medical School Paddington, London W2 IPG
L . C . ARCH~atD
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PARKH--tJRST, J. R., PETERSON, A. R. & HEIDELBERGER, C. (1973)- Breakdown of HeLa cell D N A mediated by
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activity in vitro by nick translation with DNA polymerase I. Journal of Alolecular Biology xx3, 237-25I. SAROV,I. & EECI(ER, Y. (I967). Studies on vaccinia virus DNA. Virology 33, 369-375. SoLrrHERN, E. M. 0975). Detection of specific sequences among D N A fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 5o3-518. STUDIER, V. W. 0965). Sedimentation studies of the size and shape of DNA. Journal of Molecular Biology xI, 373-39o. VILAGIN~S, R. (I970). Action du bromohydrate d'6thidium sur le d6veloppement du virus vaccinial dans les cellules en culture de rein de babouin. I. Action sur la cin6tique de d6veloppement viral. Archivf~r die gesamte Virusforschurg 3o, 59-66. WILLIAMSON,J. O. & ARCH_'.RD,L. C. (1976). The effect of canaline on some events in vaccinia virus replication. Journal of General Virology 3o, 81--89.
(Received I7 M a y I977)
