Chromatin-Like Structures Obtained After Alkaline ... - Journal of Virology

JOURNAL OF VIROLOGY, Mar. 1977, p. 1205-1209 Copyright ©D 1977 American Society for Microbiology

Vol. 21, No. 3 Printed in U.S.A.

NOTES Chromatin-Like Structures Obtained After Alkaline Disruption of Bovine and Human Papillomaviruses MICHEL FAVRE, FRANQOISE BREITBURD, ODILE CROISSANT, AND GJtRARD ORTH* Unite de Biochimie Enzymologie, Laboratoire de Pharmacologie Moleculaire, no. 147 Associg au Centre National de la Recherche Scientifique, Institut Gustave-Roussy, 94800 Villejuif, France*; and Department de Virologie, Institut Pasteur, 75015 Paris, France Received for publication 26 July 1976

Four low-molecular-weight polypeptides migrating like H2a, H2b, H3, and H4 calf liver histones were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis of highly purified preparations of bovine papillomavirus (BPV) and human papillomavirus (HPV). Complexes of these polypeptides and viral DNA were isolated by agarose-gel filtration of the alkaline disruption products of both viruses. When observed under the electron microscope, these complexes appeared as circular structures composed of nucleosomes with a diameter of about 8.0 nm interconnected by a naked DNA filament. The maximal frequency of nucleosomes per molecule was 30 for both viruses, corresponding to a condensation ratio of the viral DNA of 2.5.

Papillomaviruses and polyomaviruses are as- man plantar warts, respectively, as previously signed to the papovavirus group on the basis of detailed (4). BPV and HPV nucleoprotein comthe common structural characteristics of their plexes were obtained after agarose filtration of viral particle, but are distinct by virtue of their the alkaline disruption products of the virions genome size, their capsid diameter, and the (4). Histone preparations were obtained from molecular weight of their main structural poly- calf liver and purified according to Frearson peptide (1, 4, 15, 17). The existence of a viral and Crawford (6). Highly purified preparations core containing DNA and cellular histones may of BPV and HPV virions (2 to 4 mg/ml in 0.2 M be a further common structural characteristic sodium phosphate buffer, pH 7.4), polypeptides of papovaviruses. Complexes of viral DNA and present in the agarose column fractions, and low-molecular-weight polypeptides correspond- histone preparations (1.8 mg/ml in 0.2 M acetic ing to H2a, H2b, H3, and H4 histones (6, 12, 16) acid) were dissociated before sodium dodecyl have been isolated after alkaline disruption of sulfate (SDS)-gel electrophoresis by a 5-min insimian virus 40 (SV40) and polyoma (Py) virus cubation in boiling water in the presence of 4 M (6, 7, 11). The complexes isolated from SV40 urea, 2% SDS, and 2% 2-mercaptoethanol. SDSvirions appear in the electron microscope as polyacrylamide cylindrical gel electrophoresis circular molecules of 20 nucleosomes with a was performed as previously described (4) exdiameter of about 10 nm, interconnected by cept for the size of the gels (0.6 by 13 cm) and naked DNA filaments (9). Similar chromatin- the time of electrophoresis (16 h). Acrylamidelike structures have been found in complexes sucrose gradient slab gels were prepared in a isolated from cells lyrically infected with SV40 Hoefer apparatus (HSI Inc., San Francisco). or Py virus (2, 10). The isolation of nucleopro- The linear gradient of acrylamide (7.5 to 20%) tein complexes from bovine papillomavirus and sucrose (5 to 17.5%) and the 3% acrylamide (BPV) has been reported (4). In this note, we stacking gel were prepared as described by report that DNA-histone complexes may be iso- Maizel (13). Samples were applied with 5 ,ul of lated after alkaline disruption of particles of bromophenol blue (0.0005%) as a tracking dye. human papillomavirus (HPV) and that both After a 17-h electrophoresis (50 V), the gels BPV and HPV nucleoprotein complexes have a were stained for 1 h with Coomassie brilliant chromatin-like structure similar to that de- blue (0.25%) and destained in a methanol-acetic scribed for SV40 and Py virus (2, 9, 10). acid-water mixture (5:1:5). The molecular Full particles of BPV and HPV were purified weights of viral polypeptides were determined from bovine cutaneous fibropapillomas and hu- as described previously (4), using cytochrome c 1205

1206

NOTES

(molecular weight, 12,500), chymotrypsinogen (molecular weight, 25,000), ovalbumin (molecular weight, 45,000), and bovine serum albumin (molecular weight, 67,000) as standard proteins. Nucleoprotein complexes were prepared for electron microscopy according to the method of Dubochet et al. (3). Carbon-coated copper grids or carbon-coated self-perforated microplastic grids, prepared as described by Fukami and Adachi (8) for bright-field examination, were activated by alternating-current glow discharge in vapors of amylamine. Hundred-fold diluted samples in 0.01 M Tris-hydrochloride buffer (pH 7.9)-0.001 M EDTA were adsorbed on the carbon films for 1 min. After removal of the liquid, the adsorbed material was stained for 1 min in a 2% aqueous solution of uranyl acetate or uranyl formate. After drying, the microplastic grids were observed at 60 kV with an Elmiskop 101 Siemens electron microscope. Carboncoated copper grids were rotary shadowed with platinum-palladium (80:20) at an angle of 8° and observed under the same conditions. The nucleoprotein complexes and the DNA molecules were measured on fourfold enlargements, using a coordinatometer connected to a PDP8 digital computer; the diameter of nucleosomes was measured with a Zeiss lens. Length calibrations were made with a grating replica (F. E. Fullam, Inc., Latham, N.Y.; 54,800 lines/ inch [21,600 lines/cm]). When analyzed by SDS-electrophoresis on acrylamide cylindrical gels, highly purified BPV and HPV virions were found to have similar polypeptide compositions, i.e., a major component (VP1) with a molecular weight of about 54,000 assumed to be the capsomere subunit, three low-molecular-weight histone-like polypeptides (VP8-10), and up to six minor irregularly detected polypeptides (VP2-7) (4). When the analysis was performed on longer acrylamide gel columns (Fig. la,b), the three low-molecular-weight bands (VP8-10) were resolved into four distinct bands, as reported for SV40 and Py virus (6, 12, 16). These bands, with migration properties similar to those of the calf liver histones H2a, H2b, H3, and H4 (Fig. ic), corresponded to polypeptides with molecular weights of 16,000, 15,000, 12,500, and 11,000, which we designate as VP8 to VP11. Only three histone-like components were detected by gradient slab gel electrophoresis of viral polypeptides. However, under these conditions, the electrophoretic mobilities of the major component of HPV (Fig. ld) and BPV (Fig. le) were slightly different and corresponded to apparent molecular weights of 54,000 and 52,000, respectively, as further demonstrated by coelectrophoresis of the viral polypeptides of the two

J. VIROL.

I197.1 in qm4mmwm 40

FIG. 1. SDS-polyacrylamide cylindrical gel electrophoresis and gradient slab gel electrophoresis of SDS-dissociated papillomaviruses. Full BPV particles (1 50 pg of proteins) (a), full HPV particles (1 50 pg of proteins) (b), and calf liver histones (20 pg of proteins) (c) were dissociated and electrophoresed in the same run for 16 h on 10% cylindrical polyacrylamide gels. Full HPV particles (50 pg of proteins) (d), full BPV particles (50 pg ofproteins) (e), and the mixture of full HPV and BPV particles (25 pg of each) with standard proteins, bovine serum albumin (BSA), ovalbumin (OVA), chymotrypsinogen (CHY), and cytochrome c (CYT) (10 pg of each) (/) were dissociated and electrophoresed for 17 h on acrylamide sucrose gradient slab gels.

viruses (Fig. if. Furthermore, the resolution obtained allowed an estimation of the molecular weight of VP1' (76,000 for HPV and 65,000 for BPV) (Fig. ld,e), which was the main minor component migrating between the top of the gel and VP1; this made it unlikely that VP1' represented a VP1 aggregate, as previously suggested (4). Exposure to alkaline pH of full particles of BPV led to the disruption of virions and allowed the separation of the major component VP1 from the histone-like polypeptides that remained associated with the viral DNA. Trace amounts of minor components VP5 to -7 were found in both free and DNA-associated states (4). Similarly, when HPV virions were dissociated for 6 h at pH 10.6 and the alkaline disruption products were subjected to filtration on an agarose column, two peaks were obtained (Fig. 2).The 260-to-280-nm absorbance ratio allowed the distinction between a DNA-containing peak eluting in the void volume of the

VOL. 21, 1977

NOTES

I 0

Ec 0

CD

0 I-

0

UJ z

z 4

o

co

0

0 a)

0

eCn

0C

m

FRACTIONS

FIG. 2. Agarose-gel filtration of the disruption products of HPV virions. Full HPV particles (2 mg) were sedimented for 2 h at 100,000 x g, and the pellets were suspended in 1 ml of 0.2 M sodium carbonate buffer, pH 10.6, containing 0.01 M dithiothreitol. After a 6-h incubation at 0C, the disruption products were layered onto an agarose (A1.5M, BioRad) column (1 by 45 cm), equilibrated at 00C with a 0.2 M sodium carbonate buffer, pH 9.0, and then elated with the same buffer at a flow rate of 10 ml/h. The column was previously calibrated with T5 phage (T5), catalase (CATA) (molecular weight, 250,000), bovine serum albumin (BSA), and cytochrome c (CYT). The fractions (0.5 ml) were collected, and the absorbance at 260 (a) and 280 (E) nm was determined. Brackets indicate the fractions analyzed by electrophoresis (26 to 28; 47 to 49).

column and a protein peak that eluted slightly behind bovine serum albumin. The polypeptides present in both peaks were characterized by SDS-acrylamide gradient slab gel electrophoresis (Fig. 3). The protein peak (Fig. 3g-i) contained only VP1 (Fig. 3b), whereas the DNA-containing peak (Fig. 3d-f) consisted of the histone-like polypeptides, VP1, and trace amounts of a minor component that migrated like VP5 (Fig. 3b,c). The histone-like polypeptides constituted about 87% of the proteins in the peak fraction of the DNA-containing peak, as determined by measuring the area under the peaks obtained in a densitometric recording of the stained gels. BPV and HPV nucleoprotein complexes,

1207

characterized by a weight ratio of histone-like proteins to total protein of 84 and 87%, respectively, were observed under the electron microscope using the conditions described by Dubochet et al. (3). They appeared as circular molecules comprised of spheroid beads with a diameter of about 12 nm, corresponding to nucleosomes (9), interconnected by DNA filaments of variable length (Fig. 4a,b). This chromatin-like structure was similar to that described for nucleoprotein complexes isolated from SV40 virions or from cells lyrically infected with SV40 or Py virus (2, 9, 10). Brightfield examination of BPV and HPV complexes stained with uranyl acetate without further shadowing showed structures analogous to those described by Finch et al. (5), i.e., circular images surrounded by a dark ring and sometimes containing a central dark spot (Fig. 4c). Under these conditions, the diameter of nucleosomes was 8.3 + 1 and 8.1 ± 1 nm for BPV and HPV complexes, respectively, in agreement with the values reported for chromatin (5, 14). A maximal number of 31 and 32 nucleosomes was found for BPV and HPV complexes, respectively, with a maximal frequency of 30 nucleosomes per complex (Fig. 5). The length of the nucleoprotein complexes containing 30 nucleo-

BSA w

- vP1

OVA _

- VP5

CHY _

CYT

+

_

_

-VP8 -VP9.10

-vP11

b c d e f g h i FIG. 3. Gradient slab gel electrophoretic analysis a

of viral components obtained after agarose filtration of alkali-disrupted HPV virions. Standard proteins, bovine serum albumin (BSA), ovalbumin (OVA), chymotrypsinogen (CHY), and cytochrome c (CYT) (a), full HPV particles (100 Mg of proteins) (b), calf liver histones (20 pg of proteins) (c), and 20- uJ samples of the fractions of the agarose column (fractions 26 [d], 27 [e, peak fraction], and 28 [fi of the DNAcontaining peak, and fractions 47 [g], 48 [h, peak fraction], and 49 [ii, of the protein peak) were dissociated, electrophoresed for 16 h, and stained.

1208

J. VIROL.

NOTES

I

; ' r E t o o0 i , f *":

C

>,0 X, C

a 4 ''-''-.',,A' n XA,'' 4

.}

'0

i. ..,

-;.- A :-, lz t'...

".I -d

FIG. 4. Electron micrographs of BPV and HPV nucleoprotein complexes. (a) Pt-Pd-shadowed BPV nucleoprotein complexes; (b) Pt-Pd-shadowed HPV nucleoprotein complexes; (c) HPV nucleoprotein complex positively stained with 2% uranyl acetate; (d) Pt-Pd-shadowed HPV nucleoprotein complex and nucleosome-free HPV DNA molecule present in the same preparation. Bar indicates 0.2 gim.

somes, determined on photographs of relaxed molecules, was 1.0 + 0.1 gm for both viruses. A small proportion of form II molecules (Fig. 4d) was observed in the preparations of complexes; the length of the molecules was 2.5 + 0.1 gm for

both DNAs. From these results, a condensation ratio of about 2.5 may be calculated for the complexes containing 30 nucleosomes, in agreement with the values obtained for SV40 and Py virus complexes (2.6 and 2.7, respectively) (2).

VOL. 21, 1977

NOTES

1209

ular weight of polyomaviruses (about 3.5 x 106) and papillomaviruses (about 5 x 106) (1, 2, 17). 10

EL

5

-

0

HPV

o

CD

15

-

.0

E 10 z

5

10 20 30 Number of nucleosomes

FIG. 5. Histograms of the nucleosome number of HPV and BPV nucleoprotein complexes. Nucleoprotein complexes of HPV and BPV present in the peak fraction of the DNA-containing peak obtained after agarose filtration of the alkali-disrupted particles were prepared for electron microscopy, stained, and Pt-Pd shadowed as described in the text. Nucleosomes were counted on 114 complexes for BPV and 180 complexes for HPV.

Determination of the number of base pairs packed in a nucleosome may be inferred from the number of base pairs contained in HPV DNA and from the length of DNA contained in the nucleosome. The number of base pairs of HPV DNA has been estimated to be 8,100, from the ratio between the lengths (1.474 + 0.006) of HPV DNA form II and the replicative form of OX174 phage DNA (5,500 base pairs) (2), measured on the same grid (0. Croissant et al., unpublished data). The length of DNA contained in a nucleosome was determined from the difference between the length of naked DNA and the length of internucleosomal DNA, i.e., the difference between the length of the complexes containing 30 nucleosomes and the sum of nucleosome diameters. The value obtained, about 58 nm, corresponds to about 180 base pairs and is consistent with the number of base pairs packed in a nucleosome of SV40 and Py virus complexes (175 to 205 base pairs) (2). The results reported in this paper show that the particles of both polyomaviruses and papillomaviruses are characterized by a viral core showing a chromatin-like structure. The difference in the number of nucleosomes per complex (20 to 21 for SV40 and Py virus, 30 for HPV and BPV) reflects the difference in the DNA molec-

We gratefully acknowledge P. Sheldrick and M. Girard for critical reading of the manuscript, and M. Yaniv and J. Dubochet for helpful discussions while this work was in progress. We are greatly indebted to P. Agache for generously providing human plantar warts. We also thank C. Dauguet for technical collaboration, N. Jibard and D. Fortin for excellent assistance, and D. Cany for preparation of illustrations. This investigation was supported by the Centre National de la Recherche Scientifique through the Laboratoire Associe no. 147 (Pharmacologie Mol6culaire), and by a grant from the Institut National de la Sante et de la Recherche M6dicale (A.T.P. no. 73.4.432.18 "Enzymologie des Virus Oncogenes").

LITERATURE CITED 1. Crawford, L. V. 1963. A comparative study of polyoma and papilloma viruses. Virology 21:258-263. 2. Cremisi, C., P. Pignatti, 0. Croissant, and M. Yaniv. 1976. Chromatin-like structures in polyoma virus and simian virus 40 lytic cycle. J. Virol. 17:204-211. 3. Dubochet, J., M. Ducommun, M. Zollinger, and E. Kellenberger. 1971. A new preparation method for dark-field electron microscopy of bio macromolecules. J. Ultrastruct. Res. 35:147-167. 4. Favre, M., F. Breitburd, 0. Croissant, and G. Orth. 1975. Structural polypeptides of rabbit, bovine, and human papillomaviruses. J. Virol. 15:1239-1247. 5. Finch, J. T., M. Noll, and R. D. Kornberg. 1975. Electron microscopy of defined length of chromatin. Proc. Natl. Acad. Sci. U.S.A. 72:3320-3322. 6. Frearson, P. M., and L. V. Crawford. 1972. Polyoma virus proteins. J. Gen. Virol. 14:141-155. 7. Friedman, T., and D. David. 1972. Structural roles of polyoma virus proteins. J. Virol. 10:776-782. 8. Fukami, A., and K. Adachi. 1965. A new method of preparation of a self perforated microplastic grids and its application. J. Electron Microsc. 14:112-118. 9. Germond, J. E., B. Hirt, P. Oudet, M. Gross-Bellard, and P. Chambon. 1975. Folding of the DNA double helix in chromatin-like structures from SV40. Proc. Natl. Acad. Sci. U.S.A. 72:1843-1847. 10. Griffith, J. D. 1975. Chromatin structure deduced from a mini chromosome. Science 187:1202-1203. 11. Huang, E. S., M. K. Estes, and J. S. Pagano. 1972. Structure and function of the polypeptides in simian virus 40. I. Existence of subviral deoxynucleoprotein complexes. J. Virol. 9:930-937. 12. Lake, R. S., S. Barban, and N. P. Salzman. 1973. Resolutions and identification of the core deoxynucleoproteins of the simian virus 40. Biochem. Biophys. Res. Commun. 54:640-647. 13. Maizel, J. V. 1971. Polyacrylamide gel electrophoresis of viral proteins, p. 180-247. In K. Maramorosh and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press Inc., New York. 14. Olins, A. L., and D. E. Olins. 1974. Spheroid chromatin units (Pi bodies). Science 183:330-332. 15. Pass, F., and J. V. Maizel. 1973. Wart-associated antigens. II. Human immunity to viral structural proteins. J. Invest. Dermatol. 60:307-311. 16. Seehaffer, J. G., and R. Weil. 1974. Synthesis of polyoma virus structural polypeptides in mouse kidney cells. Virology 58:75-85. 17. Wildy, P. 1971. Classification and nomenclature of viruses, p. 38-39. In J. L. Melnick (ed.), Monographs in virology, vol. 5. S. Karger, Basel.