VIROLOGY 107, 3()6-.710 (I!%@

Virion Core-like Organization of lntranuclear Adenovirus Chromatin Late in Infection

MARTHA BROWN AND JOSEPH WEBER’

Dipartement de Microbiologic, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Qu6bec JlH 5N4 Canada

Accepted August 13, 1980

Blot-hybridization experiments following micrococcal nuclease digestion of intranuclear DNA from cells infected with adenovirus type 2, late in the infectious cycle, demonstrated that the intranuclear viral chromatin is organized in a manner similar to that of encapsidated viral chromatin in mature virions and unlike that of cell chromatin. Although a nucleosome repeat pattern was not observed with viral chromatin, a 150-base pair nuclease resistant nucleosome core DNA was nevertheless obtained. Ts3, a late mutant defective in assembly, had an intranuclear chromatin structure indistinguishable from that of WT. Results also demonstrated that the characteristic nucleosome repeat pattern of cell chromatin was not affected by adenovirus infection.

The structure of eucaryotic chromatin has been studied extensively in recent years and it is now well known that the DNA is organized into nucleosomes consisting of a DNA core 145 base pairs long associated with an octamer of histones H2A, H2B, H3, and H4 and a linker region of DNA, lo- 100 base pairs long, associated with histone Hl (1). Partial digestion of eucaryotic chroma- tin with micrococcal nuclease cleaves the DNA between the nucleosome cores result- ing in a series of fragments which are inte- gral multiples of the mononucleosome length. These can be separated by electro- phoresis in agarose gels, giving rise to a characteristic nucleosome repeat pattern (Fig. 1, f-m). SV40 chromatin has also been shown to have a nucleosome structure by virtue of the association of cellular histones with the SV40 DNA (g-4).

A study of the organization of viral DNA in purified virions of adenovirus type 2 by means of micrococcal nuclease digestion re- vealed that the viral DNA has a nucleosome structure but does not give rise to a nucleo- some repeat pattern typical of cellular chromatin t(5), Mirza and Weber, sub- mitted for publication). This is presumably due to the association of encapsidated virion DNA with core proteins V and VII (5, 6).

I To whom reprint requests should he addressed.

Adenoviruses do not contain histones. In fact histone and DNA synthesis are sup- pressed late in the infectious cycle (7). Whereas histone synthesis is dependent on continued DNA replication (8, 9), synthesis of proteins V and VII requires initiation but not continuation of DNA replica- tion (10).

Unlike encapsidated DNA, parental intranuclear viral DNA early in infection has a nuclease digestion pattern similar to that of cell chromatin (11, 12). It would appear therefore that the parental DNA loses its core proteins during uncoating (13) and acquires cellular histones, thus exhibiting a cell chromatin-like nuclease digestion pattern (11, 12). This cell chromatin-like organization is retained by the parental viral DNA even late in infec- tion (11). However, since histone synthesis is suppressed in adenovirus infected cells (7) and since encapsidated viral DNA is associated with proteins V and VT1 (6), we suggest that progeny viral DNA late in in- fection is organized by viral-coded proteins, hence its structure should be similar to that of the core. Evidence obtained from blot- hybridization experiments using ““P-labeled viral DNA probes indicates that this is in- deed the case.

Cells were infected with either WT or tsd, a mutant which synthesizes DNA but

004%6822/80/150306-05$02.00/O Copyright T 19W by Academx Press. Inc. All right, of reproduction in any form reserved.

306

SHORT COMMUNICATIONS 307

aDcdefahi lhlmn

FIG. 1. Micrococcal nuclease digestion pattern of virion chromatin and total intranuclear chromatin from infected cells. (a-e) Pentonless virions digested for (a) 0, (b) 0.5, (c) 5, (d) 10, and (e) 25 min. (f-i) Nuclei from WT infected cells digested for (f) 0.5, (g) 5, (h) 10, and (i) 25 min. (j-m) Nuclei from fss- infected cells digested from (j) 0.5, (k) 5, (1) 10, and (m) 25 min. (n) Hoe111 digest of @X174 DNA. Gel was stained with ethidium bromide and photographed under uhraviolet light. M: mononucleosome, D: dinucleosome, T: trinucleosome.

fails to form particles at 39” (14), at 100 plaque-forming units (PFU) per cell and in- cubated at 39” in medium supplemented with 2.5% calf serum and 0.8 mJ2 arginine. Nuclei prepa.rations were made 18 hr post- infection (p.:i.) by treatment of cells with lysis buffer [lo mM Tris-HCl (pH 7.9), 1 mM CaCl,, 0.3 M sucrose, 0.5% Nonidet- P40, 0.1 mlM phenylmethylsulfonyl fluoride (PMSF)] at 4” for 15-30 min. Nuclei were suspended in. digestion buffer [ 10 r&4 Tris- HCl (pH 7.9), 0.1 n-&f CaCl,, 0.3 M sucrose, 0.1 mM PMSF] and DNA concentration was measured by ultraviolet absorption at 260 and 280 nm (15). Micrococcal nuclease (Worthington Biochemicals) was added (0.03-0.05 unitipg DNA in a volume of 1.5 ml) and sarnples were incubated at 37”. Aliquots were removed after 0.5, 5, 10, and 25 min and added to tubes containing EDTA (final concentration 20 mM) and held on ice. Samples were deproteinized with Pronase and SDS then DNA was extracted with phenol and chloroform:isoamyl alcohol (24:1), and precipitated with ethanol con- taining 0.3 mh4 sodium acetate and 10 mM MgCl,. Samples were suspended in electro- phoresis buffer [40 mJ4 Tris-HCl, pH 7.6, 5 ti sodium acetate, 1 mJ4 EDTAI con- taining 10% glycerol and 0.5% bromo-

phenol blue (16), and electrophoresed in horizontal 1.4% agarose slab gels at 2.5 V/cm for 10 hr. Gels were stained with ethidium bromide (0.5 @g/ml) for 15 min and photographed using Polaroid type 57 high- speed film.

Gels were prepared for blotting to diazo- benzyloxymethyl (DBM) filters as described (17). DBM paper was prepared from amino- benzyloxymethyl (ABM) paper (Schleicher and Schuell) just before transfer as de- scribed (18).

Gels were blotted for 3 hr using 1 M sodium acetate buffer (pH 4.0). Filters not used immediately were stored at 4” in pre- hybridization buffer, which contained 50% formamide, 5x SSC [ 1 x SSC: 0.15 IM NaCI, 0.015 M sodium citrate], 5x Denhardt’s solution (19) [lx Denhardt’s solution: 0.02% (w/v) each of Ficoll, molecular weight 400,000lpolyvinyl pyrolidonelbovine serum albumin], 50 mlM sodium phosphate buffer (pH 6.5), 1% glycine and 250-500 pgiml denatured sonicated calf thymus DNA (17). “‘P-Labeled probe DNA was prepared by nick translation of purified Ad2 or cellular DNA, using DNA polymerase I (Boeh- ringer-Mannheim, 1 unit/pug DNA in total volume 100-200 ~1) in the presence of nick- translation buffer [50 mJ4 Tris-HCl (pH 7.4), 10 mM P-mercaptoethanol, 5 mJ4 MgCl*, 50 pgiml bovine serum albumin] and equal amounts of each a-“‘P-labeled dATP, dTTP, dCTP, dGTP (Amersham, 400 Ci/ mmol). The reaction was continued at 14” for 1 hr. The mixture was extracted once with chloroform and the DNA was precipitated from the aqueous phase with ethanol at -70” for 0.5-2 hr. DNA was collected by centrifugation, air dried, denatured in 100 ~1 1 N NaOH for 10 min at room tempera- ture, neutralized with 100 ~1 1 N HCI, and used immediately. “ZP-labeled DNA probes were sometimes prepared ahead of time and stored in 1 N NaOH at -20”.

Prior to hybridization, filters were sealed in a plastic bag containing prehybridization buffer and soaked at 42” for about 3 hr. The prehybridization buffer was then re- placed with a small volume of prewarmed hybridization buffer [50% formamide, 5 x SSC, 5x Denhardt’s solution, 20 rn&! sodium phosphate buffer (pH 6.5), 100 pg/

3x SHORT C’OMMUNIC’ATIONS

A 6 C obcde tahl , abcdefgh,, bidelghl

FIG. 2. Micrococcal nuclease digestion pattern of viral and cellular chromatin in nuclei of WT and tsd-infected cells. (A) Blot was hybridized with viral specific probe (b-e) nuclei from WT-infected cells digested for (b) 0.5, (c) 5, (d) 10, and (e) 2.5 min. (f-i) Nuclei from ts.1-infected cells digested for (f) 0.5, (g) 5, (h) 10, and (i) 25 min. (a, ,j) Hi,?dIII digest of Ad2 DNA. (R) Overexposure offilter A to shou the monomer in tracks (e) and (i). (C) Same blot as in A hybridized with a cell-specific probe after de- naturation of the viral probe with 0.4 X NaOH: (b-i) same as in (A).

ml sonicated denatured calf thymus DNA] (17) and the probe DNA was added. Fol- lowing hybridization at 42” for 12- 16 hr, the filters were rinsed with washing solution A (2x SSC, 0.1% SDS) from a plastic squeeze bottle, then washed with three changes (3 min each) of solution A followed by two changes (30 min each) of solution B (0.1 x SSC, 0.1% SDS) all at 42” (17). The filter was blotted dry and exposed to Kodak X- OmatR X-ray film at -70” using a Cronex Lightning-plus intensifying screen. DBM filters could be reused after dissociation of the :j’P-labeled probe DNA with 0.4 N NaOH (30-90 min at 37”).

Micrococcal nuclease digestion of infected cell nuclei 18 hr p.i. gave rise to a nucleo- some repeat pattern as seen in gels stained with ethidium bromide (Fig. 1, f-m). Blot- hybridization experiments using nick-trans- lated ‘“P-labeled viral and cellular DNA probes made it possible to detect the viral and cellular DNA sequences separately. As expected, hybridization with a cellular DNA probe yielded a repeating pattern similar to the one seen with ethidium bromide staining (Fig. 2C). Hybridization with a viral probe, on the other hand, gave rise to a smear extending from undigested DNA to the monomer region (Figs. 2A, B). Limit digestion with micrococcal nu- clease did, however, indicate the presence

of a nuclease-resistant mononucleosome in viral chromatin. The viral monomer ap- peared to be the same size but more diffuse than the cellular monomer and extended to the submonomer region. Submonomer DNA was not detected with the cellular DNA probe, indicating that under the digestion conditions used in these experi- ments, cell DNA was not digested within the nucleosome core. Thus, the submono- mer DNA seen in the gel stained with ethidium bromide (Fig. 1) must be viral DNA. Tsd-infected cells, incubated at the nonpermissive temperature, were included in the experiments to determine the nu- clease cleavage pattern of viral chromatin under conditions in which it is not en- capsidated. Figures 2A and B indicate that the cleavage patterns of viral chromatin in WT (b-e) and fs3 (f-i) infected cells were identical. The undigested viral DNA seen in extracts from WT but not fsi-infected nuclei consists of encapsidated DNA and is thus protected from nuclease digestion. To de- termine whether the cleavage pattern of intranuclear viral chromatin was the same as or different than that of encapsidated viral chromatin, pentonless virions were ob- tained by dialysis of purified visions against 5 mJ2 Tris-HCl (pH 8.1) at 4” for 16 hr and were treated with micrococcal nuclease under the same conditions as were cell

SHORT COMMUNICATIONS 309

fohi iklm

FIG. 3. Micrococcal nuclease digestion pattern of virion chromatin compared to cellular chromatin from infected cells. Same gel as shown in Fig. 1. (a-e) Hybridized with viral probe. (f-m) Hybridized with cellular probe. Tracks labeled as described in legend to Fig. 1.

nuclei. Figures 1 (a-e) and 3 (a-e) show that chromatin from mature virions, like intranuclear viral chromatin, does not dis- play a nucleosome repeat pattern but is a heterogeneous mixture of fragments rang- ing in size from undigested DNA to sub- monomer. A. diffuse viral mononucleosome is also apparent in limit digests of virion chromatin.

The results using DBM filters for blot- ting were similar to results obtained in earlier experiments with nitrocellulose filters (data. not shown), but were more reproducible. Advantages of DBM over nitrocellulose filters for blotting include the following: (a) DNA is covalently bound to DBM paper so that there is less likeli- hood of small DNA fragments being removed from the filter during washing (l7), (b) a wide size range of DNA frag- ments can be transferred more quanti- tatively to DBM filters than to nitrocellu- lose filters (17, 20, 21), (c) DBM filters can be reused after removal of the probe with 0.4 N NaOH (17, 21).

The results presented here demonstrate that (a) the organization of intranuclear viral DNA late in the infectious cycle is similar to that of encapsidated virion

chromatin and unlike that of cell chromatin, and (b) cellular chromatin is not degraded during infection (i.e., a nucleosome repeat pattern was detected by ethidium bromide staining and by hybridization with a cell- specific probe). A previous report, based on the digestion pattern of labeled DNA from infected cells following a 1-hr pulse with [“Hlthymidine late in infection, argued that intranuclear viral DNA was cleaved by micrococcal nuclease into a series of discrete fragments similar to those of cellular chromatin (5). The results obtained by hybridization, which detects viral and cellular DNA specifically, do not support this argument, however, the observation of nuclease-resistant mononucleosome-size fragment of viral DNA is consistent with our findings (5). A discussion of the nature of the viral nucleosome and possible reasons for the lack of a nucleosome repeat in viral chromatin are presented elsewhere (Mirza and Weber, submitted for publication).

As the micrococcal nuclease digestion pattern of in ~ivo viral chromatin late in infection is indistinguishable from that of virion chromatin, it is possible that the proteins protecting the in viva chromatin are the same as or similar to those in the virion. However, early in infection, the nuclease digestion pattern of viral chroma- tin appears to be like that of cellular chromatin (11, 12). This raises the ques- tion, at what point after the onset of viral DNA synthesis do progeny molecules com- plex with the viral proteins? The putative association of viral DNA with cell histones early in infection and core proteins late in infection, may simply reflect the relative availability of histones and core proteins at different times in the infectious cycle. Al- ternatively, the association of viral DNA4 with histones early in infection may be im- portant for early transcription whereas binding of viral core proteins late in infec- tion may be a prerequisite for encapsida- tion. It would be of interest to determine the organization of and proteins associated with actively transcribed and replicating molecules, and to show whether these are similar to or different from the bulk of the viral chromatin which was the subject of this report.

310 SHORT COMMUNICATIONS

ACKNOWLEDGMENTS

This work was supported by a grant from the Na- tional Cancer Institute of Canada and the Medical Research Council of Canada. J.W. is a Research Associate of the National Cancer Institute of Canada. M.B. is a Postdoctoral Fellow of the Medical Re- search Council of Canada.

REFERENCES

1. LILLEY, D. M. J., and PARDON, J. F., Ann. Rev. Gen. 13, 197-233 (1979).

2. SHELTON, E. R., WASSARMAN, P. M., and DEPAMPHILIS, M. L., J. Mol. Biol. 125, 491- 514 (1978).

3. CR~MISI, C., Microbial. Rev. 43,297-319 (1979). I. LEBOWITZ, P., and WEISSMAN, S. M., CUT-T-. Top.

Microbial. Immunol. 87, 43-172 (1979). 5. CORDEN, J., ENGELKING, H. M., and PEARSON,

G. D., Proc. Nat. Acad. Ski. USA 73, 401- 404 (1976).

6. EVERITT, E., SUNDQUIST, B., PETTERSSON, U., and PHILIPSON, L., Virology 52, 130-147 (1973).

7. TALLMAN, G., AKERS, J. E., BURLINGHAM, B. T., and REECK, G. R., Biochem. Biophys. Res. Commun. 79, 815-822 (1977).

8. ROBBINS, E., and BORUN, T. W., Proc. Nat. Acad. Sci. USA 57,409-4X (1967).

9. STEIN, G., STEIN, J., SHEPHARD, E., PARK, W.,

and PHILLIPS, I., Biochem. Biophys. Res. Commun. 77, 245-252 (1977).

10. KIT. G., and DANIELL, E.. J. Viral. 27, 74-80 (1978).

II. SERGEANT, A., TIGGES, M. A., and RASKAS, H. J., J. Viral. 29, 888-898 (1979).

12. TATE, V. E., and PHILIPSON, L., Nucl. Acid Res. 6, 2769-2785 (1979).

13. MIRZA. M. A. A., and WEBER, J., Virology 80, 83-97 (1977).

14. WEBER, J., BEGIN, M., and CARSTENS, E. B., Virology 76, 709-724 (1977).

15. DAWSON, R. M. C., ELLIOTT, D. C., ELLIOTT, W. H., and JONES, K. M., In “Data for Bio- chemical Research,” 2nd ed.. pp. 625-626. Oxford Univ. Press (Clarendon), London/New York, 1969.

16. CRAIG, E. A., ZIMMER, S., and RASKAS. H. J., J. Viral. 15, 1202-1213 (1975).

17. WAHL, G. M., STERN, M., and STARK, G. R., Proc. Nat. Acad. Sci. USA 76, 3683-3687 (1979).

18. ALWINE, J. C., KEMP, D. J., and STARK, G. R., Proc. Nat. Acad. Sci. USA 74, 5350-5354 (1977).

19. DENHARDT, D., Biochem Biophgs. Res. Com- mwn. 23, 641-646 (1966).

20. REISER, J., RENART, J., and STARK, G. R., Bio- them. Biophys. Res. Commun. 85, 1104-1112 (1978).

21. STARK, G. R., and WILLIAMS, J. G., Nucl. Acid. Res. 6, 195-203 (1979).