Vol. 29, No. 2

JOURNAL OF VIROLOGY, Feb. 1979, p. 828-832 0022-538X/79/02-0828/05$02.00/0

Parental Adenovirus Type 2 Genomes Recovered Early or Late in Infection Possess Terminal Proteins STEPHEN E. STRAUS, ALAIN SERGEANT, MICHAEL A. TIGGES, AND HESCHEL J. RASKAS5 Departments ofPathology, Medicine, and Microbiology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 Received for publication 29 August 1978

At both early (3 h) and late (18 h) times after infection of KB cells with adenovirus 2, more than 90% of parental nuclear viral genomes exist as complexes which contain terminally linked proteins. Density shift experiments employing 5bromo-2'-deoxyuridine indicate that these parental DNA molecules remain complexed with terminal proteins after DNA replication. The persistent linkage of proteins to the termini of intranuclear viral DNA suggests that these proteins have an essential role in adenovirus replication. An adenovirus DNA-protein complex is released from virions during treatment with 4 M guanidine hydrochloride (GuHCl) (11, 12). In this complex a 55,000-dalton protein is tightly, if not covalently, linked to each 5' terminus of the genome (10). To date, there have been few reported attempts to test the intracellular role(s) of the terminal proteins. Girard et al. have ascertained that some of the intracellular replicating and progeny adenovirus type 2 DNA possesses terminal proteins (6). Similar observations have been reported for simian adenovirus (SA7) intracellular DNA (5). We have examined the structure of the infecting parental genome of adenovirus type 2. To determine whether the intranuclear parental DNA is associated with terminal proteins, we have utilized a convenient assay (1, 9, 13): restriction fragments bearing terminal proteins aggregate and do not enter agarose gels. Proteolytic digestion of the aggregated complexes liberates equimolar amounts of each terminal fragment, which then migrates normally in the gel matrix. Labeled adenovirus type 2 particles were disrupted in 4 M GuHCl, and the DNA-protein complexes were purified by chromatography through Sepharose CL-2B, as reported by Coombs and Pearson (Proc. Natl. Acad. Sci. U.S.A., in press). The purified complexes were cleaved with restriction endonucleases, and the products were analyzed by electrophoresis in agarose gels. Restriction endonuclease EcoRI was employed because the relatively large size of the terminal fragments which it produces (A and C, 58.5 and 10.3% of the genome, respectively) (7) facilitates quantitation of the extent to which virion DNA bears terminal proteins. Endo RHSma I was also employed because the

relatively small size of terminal fragments J and K which it generates (2.9 and 1.0% of the genome, respectively) (8) permits more accurate localization of the proteins to the region of the DNA termini. Figure 1 shows that greater than 90% of virion DNA molecules are associated with proteins and provides the standard for comparing the results of the analysis of intranuclear viral genomes. Intranuclear parental adenovirus type 2 DNA was recovered by GuHCl treatment and GuHClCsCl gradient fractionation (11). The purified parental DNA was cleaved with endo R.EcoRI or endo R.Sma I and analyzed by agarose gel electrophoresis. Samples were obtained at early (3 h, Fig. 2A and B) or late (18 h, Fig. 2C to F) times after infection. As for virion DNA-protein complexes, greater than 90% of the labeled terminal restriction fragments remained at the tops of the gels (Fig. 2A, C, and E). Proteolytic digestion released the labeled early or late parental terminal fragents from the aggregated complexes, thereby permitting them to migrate normally (Fig. 2B, D, and F). The intranuclear DNA fragments which aggregated at the top of gels were analyzed further. DNA eluted from the first few slices of appropriate gels was digested with proteinase K and rerun on agarose gels. The DNA retained at the top of the gel shown in Fig. 2C (late nuclear complexes) was shown to represent the terminal EcoRI fragments A and C in amounts approximating their appropriate molar ratios (Fig. 3A). The late nuclear DNA retained in the first few slices of the endo R.Sma I gels (as in Fig. 2E) included the terminal fragments J and K which were enriched approximately 33-fold over the other nonspecifically trapped and trailing fragments (Fig. 3B). Similar gel profiles were ob-

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GEL SLICE NUMBER FIG. 1. Agarose gel electrophoresis of virion DNA-protein complexes cleaved with endo R EcoRI or endo R.Sma I. /52P]- or /5H]thymidine-labeled adenovirus type 2 was prepared in KB suspension cultures and purified as described elsewhere (2, 3). Labeled virions were disrupted by treatment for 5 min at 0°C with 4 M GuHCI, 0.01 M Tris (pH 7.4), 0.001 M EDTA, 0.1% Sarkosyl, and 0.001 M /3-mercaptoethanol (solution A). Virion DNA-protein complexes were purified by chromatography through a column (1 by 13 cm) of Sepharose CL-2B in solution A. The complexes were digested by endo R.EcoRI or endo R.Sma I in the presence of 2 to 5 pg of unlabeled, deproteinized adenovirus type 2 DNA by using the conditions described (3). The digested DNA was subjected to electrophoresis in cylindrical gels of 1% agarose (14). All gels were run in buffer containing 0.5 pg of ethidium bromide per ml. When viewed under UV light, the positions of all fragments could be ascertained, therebyproviding an internal markerfor each gel. Thegels were sliced, and radioactivity was determined by counting the Cerenkov radiation or scintillation in aqueous cocktails. (A) endo R -EcoRIcleaved, 3H-labeled adenovirus type 2 DNA-protein complexes. (B) endo R-EcoRI-cleaved, 3H-labeled complexes subjected to electrophoresis after digestion with 160 pg ofproteinase K per ml at 37°C for 60 min. (C) endo R-Sma I-cleaved, 3P-labeled adenovirus type 2 DNA-protein complexes. (D) endo R.Sma I-cleaved, 32plabeled complexes treated with proteinase K.

served in the analysis of aggregated restriction fragments of virion and early nuclear DNA (data not shown). These data demonstrate that parental adenovirus type 2 genomes are recoverable from early or late nuclei as DNA-protein complexes similar to those released from virions. The proportion of intranuclear genomes which behave as complexes is comparable by the present assay to the proportion observed with virion DNA (>90%). We suggest, therefore, that all of the genomes which are released from infecting virions as complexes retain that form throughout most, if not all, of the lytic cycle. To determine whether the parental DNA-protein complexes participate in replication, density shift experiments were performed. Cultures infected with 3P-labeled adenovirus type 2 were

labeled with 5-bromodeoxyuridine. At subsequent times, nuclei were lysed with 4 M GuHCl, and liberated complexes were purified by sedimentation in GuHCl-CsCl gradients. As expected, no shift in density of the labeled parental molecules occured in cells treated with 5-bromodeoxyuridine beginning at 1 h and harvested at 3 h after infection. When 5-bromodeoxyuridine was added at 6 h, an average of 42% (range of 39 to 49% in four experiments) of 32P-labeled viral DNA counts had shifted to the density position of heavy-light hybrid molecules by 18 h after infection (Fig. 4). Gradient fractions containing light-light and heavy-light hybrid density DNA were pooled (Fig. 4B). Each pool was cleaved with endo R Sma I and analyzed on agarose gels, with and without prior proteinase K digestion. The DNA in each pool behaved as

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FIG. 2. Agarose gel electrophoresis of restriction endonuclease cleaved parental DNA-protein complexes. KB cell suspension cultures were infected with 100 PFU of labeled adenovirus type 2 per cell (4). Cultures were harvested at early (3 h) or late (18 h) times after infection, washed twice with cold phosphate-buffered saline, and resuspended at a concentration of 107 cells per ml in 0.3 M sucrose, 0.01 M Tris-hydrochloride (pH 7.9), 0.001 M CaCl2, and 0.5% Nonidet P-40. Cells were lysed at 4°C with 16 to 20 (early nuclei) or 7 to 9 (late nuclei) strokes of a Dounce homogenizer fitted with a loose pestle. Phase microscopy consistently demonstrated that greater than 95% of cells were lysed with minimal breakage of nuclei. The nuclei were washed twice with the same buffer and twice again with 0.3 M sucrose, 0.001 M Tris-hydrochloride (pH 7.9), and 0.001 M CaCl2. Early or late nuclei prepared in this manner contained an average of 15% (range, 9 to 24%) of input counts. The nuclei were lysed by treatment for 5 min at 0°C in solution A. The disrupted nuclei were centrifuged in a Ti 60 rotor in a mixture of 3.026 M CsCl in solution A at 30,000 rpm, 20°C, for 66 h (11). The gradient was fractionated, and portions containing the labeled parental DNA were pooled. The DNA was digested with restriction endonucleases and analyzed on agarose gels before (A, C, and E) and after (B, D, and F) treatment with proteinase K. (A and B) 3H-labeled early nuclear complexes cleaved with endo R*EcoRI. (C and D) 32Plabeled late nuclear complexes cleaved with endo R .EcoRI. (E and F) 32P-labeled late nuclear complexes cleaved with endo R * Sma I. Electrophoresis ofearly nuclear complexes which were cleaved with endo R * Sma I yielded similar results (data not shown).

DNA-protein complexes, as already shown for virion DNA and intranuclear parental DNA (data not shown). Thus, parental DNA molecules which have replicated also possess terminal proteins. Because the light-light molecules are also predominantly in the form of DNAprotein complexes, it is apparent that their failure to replicate by 18 h is not because of the lack of terminal proteins. The present data permit a number of inferences regarding the possible role(s) of the terminal protein. Four models of the relationship between the terminal proteins and intranuclear genomes are depicted schematically in Fig. 5. Models 1 and 2 feature the removal of terminal proteins at very early stages of the lytic cycle and addition of new proteins in preparation for

packaging. Because parental adenovirus type 2 genomes are recoverable from early or late nuclei as DNA-protein complexes (Fig. 2 and 3), models 1 and 2 are excluded. Th-e density shift experiments are consistent with models 3 and 4. Model 3 outlines a process in which one end of newly replicated parental molecules lacks terminal protein. Model 4 assumes that new terminal proteins are added during or just after replication. The density shift experiment (Fig. 4) established that the parental 5' termini were complexed with protein after completion of replication. Because of the long exposure to 5-bromodeoxyuridine in our experiments, we could not study newly replicated molecules in particular and thereby discriminate between models 3 and 4.

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FIG. 3. Agarose gel electrophoresis of DNA eluted from the top few slices ofgels containing late nuclear DNA protein complexes cleaved with restriction endonucleases. DNA was recovered from gel slices with a 60 to 70% efficiency as described elsewhere (7). The DNA fragments were digested with proteinase K before rerunning. (A) DNA eluted from the top five slices of a gel equivalent to that shown in Fig. 2C. The parental DNA protein complexes had been cleaved with endo R.EcoRI. (B) DNA eluted from the top 10 slices of a gel equivalent to that shown in Fig. 2E. The parental DNA-protein complexes had been cleaved with endo R Sma I. Similar results were obtained when virion or early nuclear fragments were rerun (data not shown).

The present data do not indicate whether the proteins associated with the intranuclear parental DNA are the same 55,000-dalton molecules which were linked to the DNA in the infecting genome (10). Terminal proteins might be cleaved off, exchanged, and/or replaced with new molecules during the course of infection. We have been able to recover parental, labeled, 50,000- to 55,000-dalton proteins from purified late nuclear complexes. The efficiency and reproducibility of recovery were too variable, however, to permit quantitation. The results of previous studies may be analyzed for the predictions that they permit regarding the models of Fig. 5. Transfection studies which demonstrated marked enhancement of infectivity of adenovirus DNA bearing terminal proteins (1, 13) are consistent with all four models. They do not permit conclusions regarding the continued linkage of terminal proteins after entry of the infecting complex into the cell. Those studies which revealed the association of terminal proteins with late nuclear genomes (5, 6) did not examine the fate of the parental molecules and, therefore, remain consistent with all four models. Girard et al. reported that replicating adenovirus genomes bear terminal proteins (6). The pool of replicating parental molecules was not examined separately. Those results when combined with the present data best support model 4 and are consistent with an essential

role for the terminal proteins in adenovirus replication. This study was supported by Public Health Service grant CA16007 from the National Cancer Institute and by American Cancer Society grant VC-94D. S.E.S. was supported in this work by a Damon Runyon-Walter Winchell Cancer Fund Fellowship (DRG-112-FT) and a Public Health Service training grant (5 T01 AI00459) from the National Institute of Allergy and Infectious Disease. Cell culture media were prepared in a cancer center facility funded by the National Cancer Institute. This study was also supported by Brown & Williamson Tobacco Corp.; Larus and Brother Co., Inc.; Liggett & Myers, Inc.; Lorillard, a Division of Loews Theatres, Inc.; Philip Morris, Inc.; R. J. Reynolds Tobacco Co.; United States Tobacco Co.; and Tobacco Associates, Inc. LL

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FIG. 4. GuHCI-CsCI gradient sedimentation analysis ofparental DNA-protein complexes labeled with or without 5-bromodeoxyuridine. A 500-ml culture was infected with 32P-labeled adenovirus 2 and divided into two portions. 5-Fluorodeoxyuridine was added to one portion to a final concentration of 5 x 1(56 M at 5.5 h. 5-Bromodeoxyuridine was added to a concentration of 10'- M at 6 h after infection (15). Both cultures were harvested at 18 h. Nuclei were purified, lysed with solution A, and centrifuged to equilibrium in 3.25 M CsCl in solution A by the conditions described in the legend to Fig. 2. The density distribution of DNA from the control culture is shown in (A), and that of the sample treated with 5-fluorodeoxyuridine and 5-bromodeoxyuridine is shown in (B). Fractions 12 to 24 (containing heavylight hybrid density DNA) and fractions 27 to 35 (containing light-light density DNA) were pooled from the gradient depicted in panel B and analyzed by restriction endonuclease digestion and gel electrophoresis. Parallel gradients contained DNA-protein complexes released from 3H-labeled, 5-bromodeoxyuridine-treated adenovirus type 2 virions (heavyheavy marker) and 32P-labeled adenovirus type 2 virions (light-light marker). The brackets delineate the position of light-light markers in the presence of large amounts of cellular DNA. In the absence of cellular DNA the viral marker sedimented sharply in the position of fraction 31.

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nmIT e IfITITtETo Preparation for +M.lIT cT + + u Packaging 0rITIITI0 0nLTr 0ImnnTi T FIG. 5. Four possible models for the relationship between the terminal proteins and intranuclear parental and replicated adenovirus type 2 genomes. The infecting virion possesses proteins linked to each DNA terminus. Model 1: The infecting genome is stripped of terminal protein (0) during uncoating. Newly synthesized proteins (0) are joined to the DNA termini at the time of virion assembly. Model 2: The infecting genome retains the parental terminal proteins upon entry to the nucleus. These proteins are removed during preparation for replication and are replaced by newly synthesized proteins at the time of virion assembly. Model 3: The parental terminal proteins remain associated with the parental strands throughout replication. Newly synthesized proteins are joined to progeny strands during preparation for assembly. Model 4: The parental terminal proteins remain associated with the parental strands throughout replication. Newly synthesized terminal proteins are joined to progeny strands in close temporalproximity to replication.

We acknowledge our gratitude to David Coombs and George Pearson for advance copies of their forthcoming publication, to David Schlessinger for critical comments, to Harold Sims and Ray Barbeau for technical support, and to Diane Smerdon for assistance in preparation of this manuscript.

LITERATURE CIED 1. Chinnadurai, G., S. Chinnadurai, and M. Green. 1978. Enhanced infectivity of adenovirus type 2 DNA and a

DNA-protein complex. J. Virol. 26:195-199. 2. Craig, E. A., and H. J. Raskas. 1974. Two classes of cytoplasmic viral RNA synthesized early in productive infection with adenovirus 2. J. Virol. 14:751-757. 3. Craig, E. A., and H. J. Raskas. 1976. Nuclear transcripts larger than cytoplasmic mRNAs are specified by segments of the adenovirus genome coding for early functions. Cell 8:205-213. 4. Craig, E. A., S. Zimmer, and H. J. Raskas. 1975. Analysis of early adenovirus 2 RNA using Eco R.RI viral DNA fragments. J. Virol. 15:1202-1213. 5. Estes, M. K. 1978. Characterization of DNA-protein complexes from simian adenovirus SA7. J. Virol. 25: 917-922. 6. Girard, M., J-P. Bouche, L. Marty, B. Revert, and N. Berthelot. 1977. Circular adenovirus DNA-protein complexes from infected HeLa cell nuclei. Virology 83:

34-55.

7. McGrogan, M., and H. J. Raskas. 1977. Species identi-

fication and genome mapping of cytoplasmic adenovirus 2 RNAs synthesized late in infection. J. Virol. 23: 240-249.

8. Mulder, C., J. R. Arrand, H. Delius, W. Keller, U. Pettersson, R. J. Roberts, and P. A. Sharp. 1974. Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases Eco RI and Hpa I. Cold Spring Harbor Symp. Quant. Biol. 39:397-400. 9. Padmanabhan, R., and R. V. Padmanabhan. 1977. Specific interaction of a protein(s) at or near the termini of adenovirus 2 DNA. Biochem. Biophys. Res. Commun. 75:955-964. 10. Rekosh, D. M. K., W. C. Russell, A. J. D. Bellett, and A. J. Robinson. 1977. Identification of a protein linked to the ends of adenovirus DNA. Cell 11:283-295. 11. Robinson, A. J., and A. J. D. Bellett. 1974. A circular DNA-protein complex from adenoviruses and its possible role in DNA replication. Cold Spring Harbor Symp. Quant. Biol. 39:523-531. 12. Robinson, A. J., H. B. Younghusband, and A. J. D. Bellett. 1973. A circular DNA-protein complex from adenoviruses. Virology 56:54-69. 13. Sharp, P. A., C. Moore, and J. L. Haverty. 1976. The infectivity of adenovirus 5 DNA-protein complex. Virology 75:442-456. 14. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agaroseethidium bromide electrophoresis. Biochemistry 12: 3055-3063. 15. Vlak, J. M., Th. H. Rozijn, and F. Spies. 1976. Replication of adenovirus type 5 DNA in KB cells: localization and fate of parental DNA during replication. Virology 72:99-109.

Parental adenovirus type 2 genomes recovered early or late in infection possess terminal proteins.

Vol. 29, No. 2 JOURNAL OF VIROLOGY, Feb. 1979, p. 828-832 0022-538X/79/02-0828/05$02.00/0 Parental Adenovirus Type 2 Genomes Recovered Early or Late...
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