JOURNAL OF VIROLOGY, Aug. 1975, p. 456-458 Copyright 0 1975 American Society for Microbiology

Vol. 16, No. 2 Printed in U.S.A.

Association of Endonuclease Activity with Serotypes Belonging to the Three Subgroups of Human Adenoviruses R. G. MARUSYK,* A. R. MORGAN, AND G. WADELL Departments of Medical Bacteriology* and Biochemistry, University of Alberta, Edmonton, Alberta T6G 2E1, Canada, and Department of Virology, Karolinska Institute, Stockholm, Sweden

Received for publication 10 March 1975

Endonuclease activity has been detected in association with highly purified virions, pentons, and/or dodecons of adenovirus types 2, 3, 5, 9, 12, 15, and 16. Only single-strand scissions in substrate DNA were detected. The nuclease activity was detected by a highly sensitive ethidium bromide fluorimetric assay procedure.

Burlingham et al. (3) reported the presence of an endonuclease activity in association with human adenovirus type 2 and 12 virions and excess pool pentons. The finding of this enzyme activity was correlated to the finding of a slowly sedimenting (18S) viral DNA species in infected cells and in in vitro incubation mixtures of intact viral DNA and adenovirus type 2 and 12 virions or excess pool pentons. The adenovirusassociated endonuclease activity was reported to make only double-stranded scissions at specific sites coinciding with the guanine-cytosinerich regions of the adenovirus genome (1, 2). It was concluded by Burlingham and Doerfler (2) that the detected endonuclease activity was viral specific and virion associated. We report here the association of a single-strand scission endonuclease activity with virion-derived and excess pool viral structural components of representative serotypes from all three subgroups of human adenoviruses. We also report the use of a newly developed highly sensitive ethidium bromide fluorimetric assay (6) for detection of

isolated by adsorption to and elution from erythrocytes, can be obtained in a highly purified form (G. Wadell, Ph.D. thesis, Karolinska Institute, Stockholm, Sweden, 1970). Dodecons have been postulated to contain an internal component, the pentagon (7, 12). The signifi-

cance of this component in relation to pentonand dodecon-associated endonuclease activity was evaluated by comparison of the relative endonuclease activity in preparations of dodecons and spontaneously released dodeconderived pentons separated by rate zonal centrifugation. Preparations of adenovirus type 3 dodecons showed a pronounced endonuclease activity, but not significantly higher efficiency than the dodecon-derived pentons. Of interest was the observation that high levels of endonuclease activity were associated with the virion-derived pentons of all serotypes examined (Table 1). Burlingham et al. (3) were not able to detect endonuclease activity in any virion-derived component material. Virionderived pentons were readily obtained by dialthe adenovirus-associated endonuclease activity. ysis of highly purified virions (shown to be endoWhen highly purified adenovirus virions were nuclease negative) versus distilled water for 30 tested for the presence of endonuclease, no such min, followed by dialysis against 0.005 M activity was detectable with the assay condi- Tris-maleate buffer, pH 6.2, for 20 h at 4 C. The tions used (see legend to Fig. 1). Coded speci- dialysate was submitted to centrifugation at mens of virion-derived pentons, hexons, and 80,000 x g for 90 min, and the supernatant was DNA-containing core components, as well as used as a source of virion-derived pentons. The excess pool pentons and dodecons (naturally pellet was resuspended in 1.0 ml of a buffer occurring symmetrical aggregates of 12 pentons mixture containing 0.01 M glycine, 0.001 M from the excess pool) of several serotypes, were Tris-hydrochloride, pH 7.2, and 0.01% Triton tested, and results indicated that only virion- X-100 and subjected to 16 cycles of freezing and derived pentons, excess pool pentons, and dode- thawing. The mixture was centrifuged at 80,000 cons displayed endonuclease activity (Table 1). x g for 60 min, and the supernatant and pellet All assayed preparations of hexons, both virion were denoted virion-derived hexons and DNA derived and from the excess pool, and core core, respectively. This method of virion degracomponents were negative. dation is essentially similar to that described by Dodecons of adenovirus types 3, 9 and 15, Prage et al. (10). 456

VOL. 16, 1975

NOTES

TABLE 1. Endonuclease activity in preparations of virions and v*ion-derived and excess pool-derived pentons and dodeconsa Viral component

Endonuclease activity is expressed in terms of the presence or absence of nicked closed circular PM2 DNA substrate molecules, as determined by the fluorimetric assay and as verified by analytical ultracentrifugation, after incubation with any of the examined preparations. All adenovirus serotypes were cultivated in KB cells or HeLa cells and released from degenerating cell cultures by sodium deoxycholate treatment as previously described (5) or by several cycles of freeze-thawing. Virus particles were separated from structural components by centrifugation on discontinuous CsCl gradients (5), followed by two cycles of equilibrium centrifugation in CsCl. Virionderived pentons, hexons, and DNA-containing cores were prepared as described. All virion-derived components were obtained from purified virion preparations diluted to contain the same optical density at 260 nm per ml. As samples were identified by code numbers only, no effort was made to quantitate the actual amount of endonuclease activity present in each preparation. Excess pool pentons were isolated by anion exchange chromatography on DEAE-Sephadex A25 combined with exclusion chromatography on spherical agarose (Bio-Gel A5m). Dodecons were obtained by adsorption and elution of excess pool components to Cercopithecus aethiops or human 0 erythrocytes (a one-step, 1,200-fold concentration and purification is obtained by this procedure; Wadell, Ph.D. thesis), followed by rate zonal centrifugation on linear sucrose gradients (5 to 20% [wt/vol] in 0.067 M phosphate-buffered saline, pH 7.2) for 16 h at 42,000 x g (9). Dodecons and pentons were detected by hemagglutination and hemagglutination-enhancement assays (8, 11). Analytical sedimentation velocity centrifugations were performed at 44,000 rpm, 20 C, in a Spinco model E ultracentrifuge using Vinograd centrifugation cells. DNA (25 ul; 0.5 optical density a

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SoL......so

Endonuclease activity

........... Ad5 virion .......... Ad5 penton, virion derived ............... + ........... Ad2 virion .......... Ad2 penton, virion derived ............... + Ad 2 penton, from excess pool ............. + Ad3 dodecon ............. ........ + Ad3 penton, dodecon derived ............. + Ad9 dodecon ............. ........ + Ad9 penton, virion derived ............... + + Adl5 penton, virion derived. Adl5 dodecon ............... ...... + .......... Adl5 virion ........... .......... Ad16 virion ........... Ad16 penton, virion derived .............. + Adl6 incomplete virion .................. Adl2 penton, virion derived .............. + .......... Adl2 virion ........... Ad2 hexon, virion derived ................ Ad2 DNA core, virion derived ............-

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FIG. 1. Fluorimetric assay of adenovirus type 5 virion-derived, penton-associated endonuclease activity. The decrease in fluorescence of the blank samples after heating was due to the presence of contaminating nicked DNA. The incubation mixture consisted of 0.01 M Tris-hydrochloride, pH 7.4, 0.1 M NaCI, 0.002 M MgCI2, and 5MAg of PM2 DNA, plus a 10-MI aliquot (approximately 0.4 Mg of protein) of the virion-derived component suspension (giving an approximate DNAprotetn molar ratio of 1:1), in a total volume of 100 Ml. The reaction was allowed to take place at 37 C, with assay aliquots being removed at appropriate intervals. The endonuclease assay was carried out in a standard fluorescing solution consisting of 0.02 M K,P,, pH 12, 0.002 M EDTA, 0.5 Mg of ethidium bromide per ml (EB solution) (6). Aliquots (10 l) from the incubation mixtures were added to 2 ml of the EB solution. The fluorescence was determined relative to a blank, with excitation taking place at 525 nm and emission at 600 nm in a Turner 430 spectrofluorimeter. The fluorimetric standard consisted of 25 Ml of calf thymus DNA solution (0.234 optical density units per ml at 260 nm ml) in 2 ml of EB solution. For heat treatment (which denatures or separates the nicked strand from the closed circular strand), the solution was heated to 98 C for 2 to 3 min and cooled to room temperature. All fluorimetric readings were at 23 C. bh, Before heating; ah, after heating. Symbols: (0) Ad5 penton preparation; (0) blank. units per ml at 260 nm) was layered onto 2.83 M CsCl in 10 mM Tris-hydrochloride, pH 8.0. Markers of PM2 DNA with single nicks (by limited pancreatic DNase treatment) and double-strand nicks (endonuclease I digestion) were used (23 and 21S, respectively). Ad, adenovirus; +, endonuclease activity detected; -, endonuclease activity not detected.

458

NOTES

The assay system used for detection of adenovirus-associated endonuclease activity is based on the fluorescence enhancement of ethidium bromide bound to duplex DNA (4). By using a circular DNA substrate (PM2 phage DNA; mol wt, 6 x 106) a very sensitive assay is obtained, since a single "nick" per closed circular DNA molecule will give rise to a relaxed circular molecule which can take up more ethidium bromide (the topological constraints of superhelical DNA having been removed). The assay is made still more sensitive by working at pH 12, since nicked molecules, after heat treatment, give no fluorescence in contrast to superhelical DNA in which, upon cooling, 100% return of fluorescence is obtained because the strands cannot separate (Fig. 1). The sensitivity of the assay is such that picogram levels of nuclease can be detected (6). Adenovirus components carrying associated endonuclease activity (adenovirus 3 dodecons or adenovirus 5 virion-derived pentons) were incubated with PM2 DNA molecules in a concentration which allowed 50% nicking of the DNA molecules. The product was submitted to neutral sedimentation velocity centrifugation, after which a peak corresponding to superhelical DNA and a 23S peak corresponding to relaxed DNA circles were obtained. After centrifugation under alkaline conditions, rapidly sedimenting superhelical DNA and two peaks of equal size with S values of 24 and 27.5, corresponding to single-stranded circular and linear molecules of unit length, were observed. This would indicate that only single-stranded breaks (scissions or nicks) had occurred. These results confirm the presence of an endonuclease activity with human adenoviruses, as detected by the ethidium bromide fluorimetric assay procedure, and substantiate the probable ubiquitous nature of the endonuclease activity within the adenovirus group. Contrary to previous communications (2, 3), data accumulated in this study have indicated that, under the test conditions used, the endonuclease manifests itself through single-strand scission activity. Also in conflict with previous reports (2, 3), it has not been possible to demonstrate intact virion-associated endonu-

J. VIROL.

clease activity, whereas it has been possible to readily obtain endonuclease activity in association with virion-derived pentons. No evidence has as yet been presented for any specific biological function of the adenovirus-associated endonuclease. Extensive studies are now underway in this laboratory to determine if the endonuclease activity has a functional role in the virus-infected cell system. We wish to acknowledge the able technical assistance of B. Larson. This work was supported by research grants from the Medical Research Council, Ottawa, and the Swedish Medical Research Council.

LITERATURE CITED 1. Burlingham, B., and W. Doerfler. 1971. Three size classes of intracellular adenovirus deoxyribonucleic acid. J. Virol. 7:707-719. 2. Burlingham, B., and W. Doerfier. 1972. An endonuclease in cells infected with adenovirus and associated with adenovirions. Virology 48:1-13. 3. Burlingham, B., W. Doerfler, U. Pettersson, and L. Philipson. 1971. Adenovirus endonuclease: association with the penton of adenovirus type 2. J. Mol. Biol. 60:45-64. 4. Le Pecq, J., and C. Paoletti. 1967. A fluorescent complex between ethidium bromide and nucleic acids. J. Mol. Biol. 27:87-106. 5. Marusyk, R., E. Norrby, and U. Lundqvist. 1970. Biophysical comparison of two canine adenoviruses. J. Virol. 5:507-512. 6. Morgan, A., and D. Pulleyblank. 1974. Native and denatured DNA, cross-linked and palindromic DNA and circular covalently-closed DNA analysed by a sensitive fluorimetric procedure. Biochem. Biophys. Res. Commun. 61:436-353. 7. Norrby, E. 1966. The relationship between the soluble antigens and the virion of adenovirus type 3. I. Morphological characteristics. Virology 28:236-248. 8. Norrby, E. 1969. The relationship between the soluble antigens and the virion of adenovirus type 3. IV. Immunological complexity of soluble components. Virology 37:565-576. 9. Norrby, E., B. Nyberg, P. Skaaret, and A. Lengyel. 1967. Separation and characterization of soluble adenovirus type 9 components. J. Virol. 1:1101-1108. 10. Prage, L., U. Pettersson, S. Hoglund, K. Lonberg-Holm, and L. Philipson. 1970. Structural proteins of adenoviruses. IV. Sequential degradation of the adenovirus type 2 virion. Virology 42:341-358. 11. Wadell, G., and E. Norrby. 1969. Immunological and other biological characteristics of pentons of human adenoviruses. J. Virol. 4:671-680. 12. Wadell, G., E. Norrby, and U. Schonning. 1967. Ultrastructure of soluble antigens and the virion of adenovirus type 4. Arch. Gesamte Virusforsch. 21:234-242.

Association of endonuclease activity with serotypes belonging to the three subgroups of human adenoviruses.

Endonuclease activity has been detected in association with highly purified virions, pentons, and/or dodecons of adenovirus types 2, 3, 5, 9, 12, 15, ...
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