Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3200-3204, November 1973

Breakdown of HeLa DNA Mediated by Vaccinia (viral DNA/alkaline sucrose gradients) J. RODNEY PARKHURST, A. R. PETERSON, AND CHARLES HEIDELBERGER* McArdle Laboratory for , University of Wisconsin, Madison, Wis. 53706 Communicated by Kenneth B. Raper, July 9, 1973

ABSTRACT Breakdown of HeLa cell DNA begins within occurs as an early event in the infection process, and that 90 min after infection with vaccinia virus at a multiplicity host-cell DNA does not appear to be reutilized for the syn- of infection of 2-plaque-forming units per cell, and ends about 7.5 hr after infection. HeLa cell DNA is degraded thesis of class I viral DNA. to a uniform size of 1 to 2 X 107 daltons, as judged by MATERIALS AND METHODS alkaline sucrose sedimentation analysis. The rate of host- cell DNA degradation by vaccinia virus increased directly , Virus, and Mode of Infection. The basic tech- with the multiplicity of infection. Sedimentation pat- niques were the same as those described by Oki et al. (8). terns in neutral and alkaline sucrose gradients of viral DNA from infected cells, as well as from partially purified HeLa S3 cells and vaccinia virus (WR ) used in these virions, indicated that two size classes of DNA were pres- experiments were shown to be free of mycoplasma by a modi- ent. Class 1 DNA sediments like T4 DNA in neutral gra- fied Hayflick method (11). dients and has a molecular weight twice that of T4 DNA in alkaline gradients. Class II DNA sediments as a mole- Labeling of HeLa Cells. Logarithmic phase HeLa cells (1 to cule of lower molecular weight than T4 DNA in both types 2 X 105 cells per ml) were cultured in medium composed of of gradients. Infection of prelabeled HeLa cells with vac- Eagle's minimum essential medium for suspension culture, cinia virus did not result either in formation of trichloro- acetic acid-soluble radioactivity or, upon purification of 10% calf serum, 0.1% pluronic F18, and antibiotics (1% of the virions, radioactivity associated with class I DNA, a combination of 5000 units of penicillin and 5 mg of strepto- indicating that vaccinia virus does not reutilize HeLa mycin per ml) with 0.1 ,uCi/ml of [2-14C]dThd (54 Ci/mol) for cell DNA. 24 hr. Extracellular radioactive dThd was removed by centrif- Vaccinia virus (a DNA virus that replicates in the cytoplasm) ugation of the cells, followed by washing with phosphate- buffered saline (pH 7.3) and a second centrifugation. The inhibits the synthesis of host-cell DNA (1-3). The virions re- pelleted cells were suspended in fresh medium and were this even after ultraviolet irradiation. Hence, tain capacity grown for at least 2 hr before viral infection. the synthesis of viral DNA is not necessary for inhibition of host-cell DNA synthesis (3). Thus far, two nucleases, an Labeled Vaccinia Virions were purified essentially as de- endonuclease and an exonuclease, are among the five enzy- scribed by Fujiwara and Heidelberger (12), except that the matic activities that have been reported to be associated with cell wash and hypotonic solutions used were those described vaccinia virions (4-8). Both nuclease activities are released by Dahl and Kates (13). In brief, partially purified virus was by treatment with Nonidet (NP40) detergent (6). Incubation obtained by Dounce homogenization of infected cells, removal of these activities in vitro with tritium-labeled lambda phage of nuclei, sonication of cytoplasmic fraction, concentration DNA results in formation of mononucleotides at pH 5, or a of virus by pelleting through 35% sucrose, followed by cen- gradual reduction in the size of lambda DNA at pH 7.5 (7). trifugation of the virus once in a linear 25-50% sucrose The role of these nucleases in vivo is still obscure, but a recent gradient. report (9) stating that the virion-associated endonuclease is located within the shortly after infection may Labeling and Purification of Bacteriophage T4B01'. Bacteri- its host Escherichia coli pinpoint its site of action. Furthermore, the results of an ophage T4BO1r and B23 were generous gifts from A. W. Kozinski. Radioactive phage was grown and autoradiographic study were interpreted to suggest that the method of Kozinski was HeLa cell DNA may be degraded and also reutilized for purified by (14) and labeled with viral DNA synthesis (10), although other investigators have 2.5 1uCi/ml of [3H]dThd (>20 Ci/mol) or 0.40,uCi/ml of shown quite clearly that reutilization does not occur under [2_14C]dThd (54 Ci/mol). normal conditions (1, 8). Neutral Sucrose Gradients. 30-ml 5-20% linear sucrose The present work provides evidence that DNA from HeLa gradients containing 0.5% Na dodecyl sulfate, 0.1 M NaCl, cells is degraded to a limited extent upon infection with and 5 mM Tris HCl (pH 7.5) were prepared in Beckman vaccinia virus and delineates the kinetics of this host-cell 326825 polyallomer tubes that had been boiled in 0.10 M DNA breakdown. We will also show that this breakdown EDTA (pH 10) for 1 hr. The final pH of the gradients was 7.7. 0.1 ml of purified labeled virions [8 X 108 plaque-forming Abbreviations: MOI, multiplicity of infection; PFU, plaque- units (PFU) per ml, 80 particles per PFU] were mixed with forming units. 0.40 ml of a lysis solution containing 2 mM Tris * HCl (pH 7.5), * American Cancer Society Professor of Oncology; to whom 0.40 M NaCl, 2 mM EDTA, 2% deoxycholic acid, 4% sucrose, requests for reprints should be sent. 2% mercaptoethanol, and 1% Na dodecyl sulfate. The final 3200 Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) Vaccinia Virus Mediated Breakdown of DNA 3201

pH of the lysis solution was 7.15. The mixture was immedi- tained with a constant MOL. Fig. 3 shows the extent of deg- ately layered on the top of the gradients and incubated for 15 radation of DNA in cells infected for 3 hr with increasing min at room temperature before sedimentation was begun. MOI. The sedimentation profile of DNA from cells infected Gradients were centrifuged at 22,500 rpm for 4 hr at 120 in a with an MOI of 6 (25 particles per PFU) was similar to that Spinco SW25.1 rotor in a model L ultracentrifuge. Fractions from uninfected cells. However, as the MOI increased, the (about 33) of 0.9 ml were collected from the bottom of the amount of DNA sedimenting as the control (155 S) decreased gradient directly into scintillation vials. with a concomitant increase in the formation of the 41S DNA. HeLa DNA Alkaline Sucrose Gradients. A published method (15) was The almost complete degradation of (155 S) used after modification. 30-ml 5-25% linear alkaline sucrose gradients were prepared containing 1.0 M NaCl, 1 mM EDTA, and 60 mM Na p-aminosalicylate with a final pH of 12.5. Isolated vaccinia virions or 1 to 3 X 105 HeLa cells were lysed for 30 min on top of the gradient, often together with reference bacteriophage T4, in an 0.4-ml layer containing 0.5 M NaOH and 5 mM EDTA. The gradients were centrifuged C.E at 24,500 rpm for 3 hr at 200 in an M-60 International ul- -JU tracentrifuge with an SB110 rotor. Fractions (about 33) of 4 0.9 ml were collected from the bottom of the gradients by 0 insertion of a stainless steel tube. Trichloroacetic acid-insol- I- uble material was collected on Gelman Type A glass-fiber filter discs, and the radioactivity was determined by scintilla- $I- tion counting. Recovery of greater than 90% of the radio- 5: activity applied was obtained in these gradients. 0 Centroids: Calculation of S Value and Molecular Weight. 0 The molecular weight and sedimentation coefficients for the cr DNA sedimented in neutral and alkaline sucrose gradients were calculated by the method of Studier (16). For some ex- periments, the centroids were calculated for the sedimenta- tion profiles according to the following formula: Centroid = 35 Enifi/Eni where ni is the net level of radioactivity in the ith fractionf. RESULTS Kinetics of Breakdown of HeLa Cell DNA in Vaccinia- Infected Cells. The majority of DNA from uninfected HeLa cells sedimented in a band with its peak at fractions 9-11 of E the alkaline sucrose gradients (Fig. 1A and B). The sedimenta- 06 tion constant calculated for the peak of this band was 155. -J In cells infected with vaccinia virus for 90 min, the fraction DNA in this was reduced half and of sedimenting region by 0I- the quantity of DNA with peaks at about fractions 19 (95 S) and 27 (41 S) increased concomitantly (Fig. 1A). A progressive time-dependent increase in the fraction of 41S DNA was ob- 5:I- served up to 7.5 hr after infection (Fig. 1B), but the sedimen- C.) 4 tation profile of DNA from cells infected for 22.5 hr remained 0 0 the same as at 7.5 hr after infection. No acid-soluble radio- 4 activity was found in infected HeLa cells or medium up to 24 hr after infection (data not shown), thus confirming the findings of Kit and Dubbs (1). Sedimentation of uninfected HeLa cells labeled both with radioactive choline and leucine demonstrated that these precursors were not recovered in the |wtXX Ae B DNA peak. 0 10 20 30 35 The centroid value, which includes all the fractions of a FRACTION NUMBER [TOP) sedimentation profile, may be used to express the relative FIG. 1. Alkaline sucrose sedimentation profiles of HeLa cell proportions of all species of DNA present (15). The centroids DNA labeled for 24-28 hr with 0.10 ,uCi/ml of [14C] dThd (54 Ci/ obtained from three separate experiments are graphed to mol). The cells were washed and resuspended in fresh medium for show that the conversion of the DNA sedimenting at 155 S 2 hr and infected with a multiplicity of infection (MOI) of 2 to DNA sedimenting at 41 S is virtually complete within 8 for (A) 0 hr (control; no infection), *; 1.5 hr, 0; 2.5 hr, X. (B) hr after infection (Fig. 2). 0 hr (control; no infection), 0; 4.5 hr, 0; 7.5 hr, A; 22.2.5 hr, X. The superimposed arrows at 155 S, 95 S, and 41 S represent Effect of Increasing Multiplicity of Infection on HeLa Cell calculations. The arrow at 71 S represents the peak position of DNA Breakdown. The data shown in Figs. 1 and 2 were ob- T4 bacteriophage DNA. Downloaded by guest on September 27, 2021 3202 : Parkhurst et al. Proc. Nat. Acad. Sci. USA 70 (1973)

120 analyzed on alkaline or neutral sucrose gradients, reproducible bimodal sedimentation profiles of DNA were obtained (Figs. 4 and 5), showing that two DNA species are present. The 100 0 most rapidly sedimenting species has been arbitrarily named - class I and the slower sedimenting, class II DNA. The peak 0 molecular weight of class I vaccinia DNA 0 80 is 1.4 to 1.6 X 108 daltons in both alkaline and neutral gradients. This value _O indicates that class I vaccinia DNA is resistant to complete 0- 60 denaturation in alkali, which is consistent with Berns and 4._0 c Silverman's suggestion that the vaccinia contains at e least one crosslink (17). Class II DNA is composed of frag- c00) 40 0a) ments with an apparent molecular weight of about 107 in neutral as well as alkaline gradients. When purified vaccinia virions were incubated with DNase under appropriate condi- 20 tions followed by lysis and sedimentation in alkaline sucrose gradients, class I viral DNA sedimented as previously ob- 0. served, while class II sedimented at the top of the gradients, 0 10 20 25 indicative of DNase sensitivity. Hours after infection A comparison of the degradation patterns seen in Figs. 1 and 3 with the sedimentation profiles of viral DNA in Fig. 4 sug- FIG. 2. Centroid position against hours after infection of ['4C]dThd-labeled HeLa cells by vaccinia virus. These data rep- gests that there is little or no reutilization of HeLa DNA by resent a replot of Fig. 1A and B and also include times of in- vaccinia virus. In order to test more rigorously whether the fection not previously mentioned. Times after infection at an observed degradation of HeLa cell DNA was accompanied MOI of 2 were: 0.2, 0.5, 1.0, 1.5, 2.5, 4.5, 7.5, 11.5, 18.0, and by its reutilization for viral DNA synthesis, we labeled the 22.2 hr. The standard error of the mean (SEM) for these experi- cells for 24 hr with 0.1 ,uCi/ml of [14C]dThd. After 24 hr, the ments is shown. cells were washed thoroughly to remove unincorporated radioactive dThd and allowed to grow in fresh medium for 2 by 3 hr after infection at an MOI of 24 implies that the onset hr before infection. Before infection the specific activity of of the degradative process is an early event in infection. the HeLa cell DNA was 5.3 X 104 dpm/,gg. 22 hr after infec- tion, virions were partially purified from the cells and sub- HeLa Cell DNA Is Not Reutilized by Vaccinia Virus. When sequently lysed and sedimented in neutral and alkaline partially purified ['H]dThd-labeled virions were lysed and

10 t 155S 71S 41S IOIS 71S 27s 10r

8 8 E E CL 6-J -j o

4 4 l PI O o 2030 3

tr 2 2

0 10 20 30 35 0 10 20 30 35 FRACTION NUMBER [TOP] FRACTION NUMBER [TOP] FIG. 4. Alkaline sucrose sedimentation of ['H]dThd-labeled FIG. 3. Alkaline sucrose sedimentation profiles of ['4C]dThd- DNA obtained from partially purified vaccinia virions after labeled DNA from HeLa cells infected with vaccinia virus for lysis. 1 hr after infection of HeLa cells with vaccinia virus, 3 hr at MOI of 0, (0); 6, (X); 12, (A); and 24 (V). The super- [3H]dThd was added to the growth medium. Mfter an additional imposed arrows at 155 S and 41 S represent calculations. The 22 hr, the tritium-labeled virions were purified. The superimposed arrow at 71 S represents the peak position of T4 bacteriophage arrows at 101 S and 27 S represent calculations. The arrow at 71 S DNA. shows the peak position of marker T4 bacteriophage DNA. Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Vaccinia Virus Mediated Breakdown of DNA 3203

gradients. No radioactivity was found in the region corre- sponding to class I viral DNA, showing that HeLa cell DNA is not reutilized by vaccinia virus for the synthesis of this class of DNA. A small amount of radioactive DNA was found near the tops of these gradients, which sedimented in the region of degraded HeLa cell DNA and class II DNA. This radioactivity, 310 dpm/,ug DNA, represents a reutilization of E 0.01% of the total HeLa cell DNA, but had a specific activity -J 5.9% of that of the HeLa cell DNA. There is not sufficient 4 information from these experiments to conclude whether there - is a small amount of reutilization of HeLa cell DNA for the 0 production of class II DNA, or whether degraded fragments of HeLa cell DNA are partially encapsulated by vaccinia virus.

DISCUSSION 0 The breakdown of HeLa cell DNA early after infection with 4 vaccinia virus implies that either a viral enzyme (carried by the virion or synthesized early after infection) or the induction and/or activation of a cellular enzyme is responsible for this degradation. Our data do not allow us to distinguish conclu- sively between these possibilities. A recent report (9), indicat- ing that a vaccinia virion-associated endonuclease is present 10 20 30 35 within the cell nucleus shortly after infection, lends support to FRACTION NUMBER (TOP] the hypothesis that the mechanism of the degradation of host- FIG. 5. Sedimentation profile of ['H]dThd-containing viral cell DNA may involve the release of a virion-associated en- DNA obtained from lysis of partially purified vaccinia virions on zyme capable of entry into the cell's nucleus, which then de- neutral sucrose gradients. Sedimentation was as described in grades the host-cell's DNA. Methods. The arrow at the top represents the peak position of Vaccinia virus appears to be unique in the sense that it is the [I4C]dThd-labeled marker T4 DNA; in this system T4 has a only DNA virus known to degrade host-cell DNA early in sedimentation constant of 62 S. productive infection. Two other whose DNA is within the and replicated nucleus (Epstein-Barr polyoma) of class I viral DNA. also break down host-cell DNA, but under different conditions Also, virions purified from HeLa cells from vaccinia. labeled for 24 hr with ['4C ]dThd before infection do not con- Infection of Raji cells (a nonvirus-producing cell line of tain 4C-labeled class I viral DNA. Burkitt's lymphoma) by Epstein-Barr virus results in Other investigators have found that DNA from vaccinia degra- virions has an apparent molecular weight of 1.5 X dation of cellular DNA (18). Thus far, we are not aware of 108 (19, 20), similar findings in cell lines in which productive infection by to which class I DNA corresponds. Hence class I DNA, which Epstein-Barr virus occurs. Also, investigators (19) working survives DNase treatment of purified vaccinia virions, must be with a polyoma virus-infected cell line demon- the viral genome. Class II DNA, which does not survive mouse-embryo DNase treatment of strated that degradation of host-cell DNA occurs late in infec- virions, has not previously been reported. tion and only after its replication. The size of this degraded The invaluable technical assistance of Mrs. Virginia Garcia and cellular DNA was reported to be about the same as that of the Miss Martha Weiss is greatly appreciated. The generous help of Dr. W. Szybalski and Dr. Karin Carlson in preparation of T4 viral DNA. phage is acknowledged. The constructive suggestions of Drs. Neither the mechanism of host-cell DNA degradation by Howard Temin and Duard Walker in the preparation of the vaccinia virus nor the significance of the resulting fragments is manuscript are gratefully acknowledged. This work was supported known. The onset of degradation is correlated in time with the in part by Grants CA-7175 and CRTY-5002 from the National first detectable viral DNA synthesis, and degradation is com- Cancer Institute, National Institutes of Health. plete by the time encapsulation of viral DNA commences (2). 1. Kit, S. & Dubbs, D. R. (1962) "Biochemistry of vaccinia- Whether or not these correlations in time are coincidental infected mouse fibroblasts (strain L-M) II. Properties of remains to be determined. the chromosomal DNA of infected cells," Virology 18, 286- 293. It has been demonstrated that vaccinia virus does not 2. Joklik, W. R. & Becker, Y. (1964) "The replication and reutilize HeLa DNA to synthesize viral DNA under normal coating of vaccinia DNA," J. Mol. Biol. 10, 452-474. conditions (1), although reutilization of host-cell DNA does 3. Jungwirth, C. & Launer, J. (1968) "Effect of poxvirus occur when cells are intranuclearly irradiated with tritiated infection on host cell deoxyribonucleic acid synthesis," thymidine and then infected with vaccinia virus In that J. Virol. 2, 401-408. (8). 4. Pogo, B. G. T. & Dales, S. (1969) "Two deoxyribonuclease special case the extent of reutilization was correlated with the activities within purified vaccinia virus," Proc. Nat. Acad. specific radioactivity of the host-cell DNA and the consequent Sci. USA 63, 820-827. appearance of a DNase with a pH optimum of 9.2 (8). Two 5. Jungwirth, C., Launer, J., Dombrowski, G. & Horak, I. lines of evidence in the present work support the finding that (1969) "Characterization of deoxyribonucleases induced by poxviruses," J. Virol. 4, 866-871. vaccinia virus does not reutilize host-cell DNA. Breakdown of 6. Schwartz, J. & Dales, D. (1971) "Biogenesis of poxviruses: HeLa cell DNA does not result in the appearance of an intra- Identification of four enzyme activities within purified cellular DNA species of molecular weight comparable to that Yaba tumor virus," Virology 45, 797-801. Downloaded by guest on September 27, 2021 3204 Cell Biology: Parkhurst et al. Proc. Nat. Acad. Sci. USA 70 (1973)

7. Aubertin, A. & McAuslan, B. R. (1972) "Virus-associated 14. Kozinski, A. W. (1961) "Fragmentary transfer of 32p_ nucleases: Evidence for endonuclease and exonuclease labeled parental DNA to progeny phage," Virology 13, activity in rabbitpox and vaccinia viruses," J. Virol. 9, 124-134. 554-556. 15. Peterson, A. R., Fox, B. W. & Fox, M. (1973) "Alkaline 8. Oki, T., Fujiwara, Y. & Heidelberger, C. (1971) "Utiliza- sucrose sedimentation studies of DNA from P3851 lym- tion of host-cell DNA by vaccinia virus replicating in phoma cells treated with difunctional alkylating agents," HeLa cells irradiated intranuclearly with tritium," J. Biochim. Biophys. Acta 299, 385-396. Gen. Virol. 13,401-413. 16. Studier, F. W. (1965) "Sedimentation studies of the size 9. Pogo, B. G. T. & Dales, S. (1973) "Direct effect of penetrat- and shape of DNA," J. Mol. Biol. 11,373-390. ing vaccinia particles on host DNA polymerase activity," 17. Berns, K. I. & Silverman, C. (1970) "Natural occurence of Fed. Proc. 32, 842 abstr. cross-linked vaccinia virus deoxyribonucleic acid," J. Virol. 10. Walen, K. H. (1971) "Nuclear involvement in poxvirus 5,299-304. infection," Proc. Nat. Acad. Sci. USA 68, 165-168. 11. Hayflick, L. (1965) "Tissue cultures and mycoplasmas," 18. Pagano, J. S. & Nonoyama, M. (1972) "Replication of Tex. Rep. Biol. Med. 23, 285-303. viral deoxyribonucleic acid and breakdown of cellular 12. Fujiwara, Y. & Heidelberger, C. (1970) "Fluorinated deoxyribonucleic acid in Epstein-Barr virus infection," pyrimidines XXXVIII. The incorporation of 5-trifluoro- J. Virol. 9, 714-716. methyl-2'-deoxyuridine into the deoxyribonucleic acid of 19. Ben-Porat, T. & Kaplan, A. S. (1967) "Correlation between vaccinia virus," Mol. Pharmacol. 6, 281-291. replication and degradations of cellular DNA in polyoma 13. Dahl, R. & Kates, J. R. (1970) "Intracellular structures virus-infected cells," Virology 32, 457-464. containing vaccinia DNA: Isolation and characterization," 20. Sarov, 1. & Becker, Y. (1967) "Studies on vaccinia virus Virology 42,453-462. DNA," Virology 33, 369-375. Downloaded by guest on September 27, 2021