Vol. 20, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, OCt. 1976, p. 9-13 Copyright © 1976 American Society for Microbiology
Origin of Polyoma Virus-Associated Endonuclease P. ROUGET,' A. PARODI,2 D. BLANGY,' AND F. CUZIN3*
Departement de Biologie MolMculaire, Institut Pasteur, Paris, France
Received for publication 26 April 1976
Polyoma virus particles purified from infected cells, but not from the culture medium, exhibited an endonuclease activity distinct from the serum contaminant recently described. This endonuclease cleaved form I polyoma DNA once only per molecule, at one of three possible sites corresponding to the known adenosine-ribosylthymine-rich regions of the molecule.
It has been recently demonstrated by McMillen et al. (7) that polyoma virus particles, purified from cell lysates, carry over, through the isopycnic step of the virus purification, a contaminant endonuclease from the culture serum. We have mentioned the same observation in a previous report and have shown that this contamination can be avoided (10). At least with the large plaque strain of polyoma propagated in our laboratory, preparations of purified virions that have not been exposed to the culture medium exhibit an endonulease activity that is different in many of its properties from the serum enzyme(s).
particles was collected from the gradient, and the dialyzed virus suspension was used as a source of enzyme without further disruptive treatment. CsCl equilibrium gradients were centrifuged for 24 to 36 h to collect the contaminant protein as a discrete band, which is not possible at earlier times of centrifugation, as in the procedure of McMillen et al. (7). Viral DNA. Polyoma form I DNA was extracted according to Hirt (6) from primary mouse kidney cells 30 h after infection and purified as previously described (3). Form I DNA was further separated from more slowly sedimenting defective molecules by an additional sucrose gradient velocity centrifugation as described below. To check the homogeneity of DNA preparations, samples were digested with Eco Rl, denatured in NaOH (final pH, 12.8) at room temperature, renatured at pH 8.7 in 50% formamide at 25°C for various times, mounted for electron microscopy using the formamide technique (4), and examined as described below. The absence of heteroduplex loops in these preparations was taken as a criterion of homogene-
MATERIALS AND METHODS Cell lines and viruses. Primary mouse kidney cells (12) were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Polyoma virus of large plaque type was originally obtained from M. Vogt. Polyoma was propagated either in baby mice according to Winocour (12) or in primary mouse kidney cell cultures infected in vitro at low multiplicity (less than 0.1 PFU/cell). Virus purification. Virus was purified from infected cells by either one of two methods. (i) Cell debris and free virus were concentrated from the whole lysate, including the medium, by high-speed centrifugation (2 h, 80,000 x g), suspended in Dulbecco TD buffer (11), frozen and thawed quickly three times, treated with neuraminidase (Sigma), and subjected to two successive cycles of isopycnic centrifugation according to Crawford (2). In method (ii), the medium was discarded, and cells were collected, washed by low-speed centrifugation, and disrupted by freezing and thawing. After neuraminidase treatment, virus was banded at equilibrium as in method (i).
ity. Enzymes. Eco Rl endonuclease was purified according to R. N. Yoshimori (Ph.D. thesis, University of California, San Francisco, 1971). Electrophoretically pure pancreatic DNase was purchased from Worthington, and neuraminidase (Vibrio cholerae filtrate) was from Sigma. Enzyme assays. The reaction mixture for the polyoma-associated endonuclease contained 10 mM Tris-hydrochloride, pH 7.4, 5 mM MgCl2, 5 mM CaCl2, 1 to 10 ,ug of DNA per ml, and 5 to 10 ,g of viral protein per ml. They were incubated at 37°C, and the reaction was stopped with an excess of EDTA. The enzyme activity was determined either by the filter binding method (3) or by sucrose gradient sedimentation as described below. Incubations with Eco * Rl endonuclease were carried out according to Morrow and Berg (9). Sucrose gradient centrifugations. DNA samples (0.1 ml) were layered on top of 5 to 20% sucrose gradients containing either 1 M NaCl, 10 mM EDTA, and 10 mM Tris-hydrochloride, pH 7.4 (neutral gradients), or 0.2 N NaOH and 0.8 M NaCl (alkaline gradients).
In both cases the band corresponding to the full I Present address; Institut de Recherches Scientifiques sur le Cancer, 94800 Villejuif, France.
2 Present address: Instituto de Investigaciones Bioquimicas, Fundacion Campomar, Obligado 2490, Buenos Aires, Argentina. 3Present address: Centre de Biochimie, Universite de Nice, 06034 Nice, France.
ROUGET ET AL.
Gradients were centrifuged at 48,000 rpm for 4 to 6 h at 200C in Beckman SW50.1 or SW56 rotors. Electron microscopy. DNA molecules were spread on collodium-coated copper grids by the formamide technique (4), shadowed with Pd-Pt, and examined using a Siemens Elmiskop 101 microscope. DNA molecules were measured with a laboratorymade coordinatometer connected to a PDP-8 digital computer. Calibration of the microphotographs was made with a carbon grating replica (EF Fulham Inc., 54,800 lines/inch).
RESULTS AND DISCUSSION Virions prepared by either method (i) or (ii) exhibited a nicking activity when tested on polyoma form I DNA (Fig. 1 and 2). The end product of the reaction was, however, different in both cases. Using virions purified from the whole lysate, including the medium [method (i)], incubating the reaction mixture three times longer than required for a complete disappearance of form I DNA led to conversion of this DNA into fragments of less than one genome length after denaturation (Fig. 1). When analyzed by neutral sucrose gradient centrifugation or by electron microscopy, this DNA appeared to be primarily made of form II circular molecules (data not shown). These results confirm and extend those presented by McMillen et al. (7), indicating the presence in the virion preparation of an enzyme activity introducing single-strand nicks at multiple sites on form I polyoma DNA. When the same experiment was done using
virions prepared from the cell fraction [method (ii)], the pattern obtained was completely different. Even after prolonged incubation, or adding a large excess of virion protein, no DNA fragments shorter than a unit-length polyoma strand appeared on the gradient (Fig. 2). The end product was in fact identical to linear molecules produced by Eco * Rl cleavage ofpolyoma DNA, both by its sedimentation properties (Fig. 2) and by its length, as measured by electron microscope analysis (10). The enzyme present, in this type of preparation therefore appears to be different from the one found in virions prepared by method (i). It cleaves polyoma DNA only once per molecule, and cleavage occurs on both strands at sites less than 4 nucleotides apart (10). By assaying in the same way endonucleases present in fetal calf serum, we could not detect the appearance of a unit-length double-stranded linear product. The two types of activities could be further distinguished by other criteria. They exhibit different apparent Km values for polyoma DNA and different heat inactivation kinetics (data not shown). The enzyme cleaving polyoma DNA only once was shown to be highly Ca2+ dependent (10), whereas that from the serum was not activated by calcium ions (7; our unpublished data). We used the term "polyomaassociated endonuclease" for the enzyme found in virions which have not been exposed to the culture medium. The endonuclease-specific activity of virion
0aII 0Gww 0ml 3in0 Ii 30min _9_m_ j
TOP_ FRACTION NUMBER FIG. 1. Cleavage of polyoma form I DNA in the presence of virions prepared by method (i). 3H-labeled polyoma form I DNA was incubated in the presence of virions obtained by method (i). Aliquots were taken after incubation for 0, 30, and 90 min and sedimented in alkaline sucrose gradients as described. Arrows indicate the position of polyoma form I DNA cleaved by Eco Rl sedimented in a separate tube. (Insert) Kinetics of cleavage estimated by the filter binding assay in the presence (0) or in the absence (A) of added virions. Ordinate: nicked DNA (percentage).
VOL. 20, 1976 .ft
Al A0mmin Neutral -0..-
r 8) 5Imin Neutrol
30 40 500 20 10 20 10 30 40 50 0 _--oOToM Froction Number TOP-. .._0TOU Froction Number TP-. 8
30 40 50 0 10 20 30 40 50 0-SOTTOM Fraction Number TOP.. -*TTOM Fraction Number TOP*. 10
E4 0o 2-
50 20 30 40 Fraction Number TOPw
FIG. 2. Cleavage of polyoma form I DNA in the presence of virions prepared by method (ii). 3H-labeled polyoma form I DNA was incubated with virions prepared by method (ii) (see Materials and Methods) under standard conditions. Portions
taken at various times and sedimented in neutral (A, B, C)
(E, F, G) sucrose gradients as described. Eco RI-cleaved polyoma [32P]DNA was added as an internal standard; 49 to 50 fractions were collected in each case. Fractions not shown had no detectable radioactivity. Arrows indicate the position of superhelical (I), relaxed circular (II), and linear (III) DNA. Kinetics of cleavage were the same as in Fig. 1.
preparations obtained by method (ii) was found one preparation to the other. Using virions with low or undetectable activity, a significant increase was observed to be variable from
when the assay was performed in the presence of sodium deoxycholate (Fig. 3). This result suggests that unmasking of a virus-bound enzyme, which may occur by spontaneous disrup-
ROUGET ET AL.
the double digestion were further analyzed by electron microscopy measurements. Five populations appeared on the histogram (Fig. 5); their fractional lengths were 0.61 and 0.43, 0.78 and 0.20, and 0.96. Due to their small size, the molecules of class 0.04, which one would expect to find complementary to those of 0.96, could not be clearly detected on the electron microscope preparations. The results indicate that the polyoma-associated endonuclease attacks form I polyoma DNA at three sites located at about 0.04, 0.20, and 0.40 genome lengths from the Eco * Rl site. These values again correspond to those of the A-T-rich regions of polyoma DNA identified by T4 gene 32 protein binding (8, 13, 14) and by Aspergillus Si endonuclease cleavage (5). The polyoma-associated endonuclease, therefore, recognizes on polyoma form I DNA the partially melted A-T-rich regions.
3-0 #~~~~~~~~ + Virions "
Z 2.0 z
FIG. 3. Enhancement of endonuclease activity in the presence of sodium deoxycholate. Conversion of form I polyoma DNA into relaxed structures was followed by the filter binding assay (3) as a function of time. All tubes contained, in a total volume of 0.1 ml: Tris-hydrochloride buffer, pH 7.4, Ca2+ and
Mg2+ as indicated in Materials and Methods, and 0.08 pg of 3H-labeled simian virus 40 form I DNA (50,000 cpm/pg). Part of the tubes were supplemented with sodium deoxycholate (DOC at a final concentration of2 x 10-3 M. Reaction was started by adding polyoma virions purified by method (ii) (2.8 pg of protein per tube) and the same amount of buffer in duplicate tubes with and without sodium deoxycholate (control).
tion of virions, may also be enhanced by detergent treatment. We have shown that this enzyme cleaves simian virus 40 DNA at one of two possible sites that correspond to the known adenosine-ribosylthymine (A-T)-rich regions of the DNA (10). The same experiment was done with polyoma DNA. Form I DNA was first converted into linear molecules by the virion-associated enzyme and thereafter submitted to extensive cleavage by Eco Rl. As shown in Fig. 4, the velocity sedimentation pattem of the doubly cleaved products indicated at least four populations of DNA molecules. Their average genome lengths, approximately estimated from the Abelson-Thomas equation (1), were 0.87, 0.62, 0.33, and less than 0.10. However, this evaluation was approximate, and each of the peaks or shoulders may represent several populations. For a more precise estimation, the products of
x z 0
- 10 z n
24 I 5 10 IS2 5 50 5540 FRACTION NUMBER TOP-0
FIG. 4. Sedimentation pattern of the products of the double digestion of polyoma DNA with the polyoma-associated and Eco R1 enedonucleases. Superhelical DNAs were extensively cleaved by the polyoma-associated endonuclease, and linear products were isolated on neutral sucrose gradients, ethanol precipitated, and dissolved in 10 mM Tris-hydrochloride buffer, pH 7.4-1 mM EDTA. The DNA obtained was exhaustively incubated with (0) or without (a) Eco -R1 enzyme and run on neutral sucrose gradients; 52 fractions were collected. Fractions not shown had no detectable redioactivity. Arrows indicate the populations discussed in the text. The difference between the heaviest population of the doubledigested products and the unit-length linear DNA was ascertained in similar gradients where the former sample was co-centrifuged with Eco -Rl-treated, 32P-labeled polyoma DNA.
VOL. 20, 1976
Cancer, and Fondation pour la Recherche Medicale Fran-
LITERATURE CITED 32 w
1. Abelson, J., and C. A. Thomas. 1966. The anatomy of
,,, 24 0
2 20 0
0.75 1.00 1.50 1.25 ABSOLUTE LENGTH (pm)
FIG. 5. Electron microscopy analysis of the products of the double digestion of polyoma DNA with
polyoma-associated and Eco R1 endonucleases. The double digestion was carried out as described in the legend to Fig. 4. Products were analyzed by electron microscopy analysis as indicated in Materials and Methods.
The polyoma-associated endonuclease therefore appears to be distinct from serum contaminants. Whether it is a cellular or a viral enzyme and whether it plays a significant role in the virus cycle remain to be established. ACKNOWLEDGMENTS We thank 0. Croissant for help and advice on the electron microscope analysis. The technical assistance of C. Dauguet, C. Maczuka, and N. Montreau is gratefully acknowledged. While this work was carried out, one of us (A. P.) was first an Eleanor Roosevelt Fellow of the International Union Against Cancer and later a John Simon Guggenheim Memorial Foundation Fellow. The work was supported by grants from the Centre National de la Recherche Scientifique (ATP "Differenciation Cellulaire" and ERA ), Institut National de la Sante et de la Recherche Medicale (AT "Enzymologie des Virus Oncogenes"), Commissariat a l'Energie Atomique, Ligue Nationale Frangaise contre le
the T5 bacteriophage molecule. J. Mol. Biol. 18:262291. Crawford, L. V. 1969. Purification of polyoma virus, p. 75-81. In K. Habel and N. P. Salzmnan (ed.), Fundamental technics in virology. Academic Press Inc., New York. Cuzin, F., P. Rouget, and D. Blangy. 1973. Endonuclease activity of purified polyoma virions, p. 188-201. In L. G. Silverstri (ed.), Possible episomes in eukaryotic cells. North Holland Publishing Co., Amsterdam. Davis, R. N., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, p. 413428. In S. P. Collowick and V. P. Kaplan (ed.), Methods in enzymology, vol. 21B. Academic Press Inc., New York. Gennond, J. E., V. M. Vogt, and B. Hirt. 1974. Characterization of the single-strand-specific nuclease S1 activity on double-stranded supercoiled polyoma DNA. Eur. J. Biochem. 43:591-00. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26: 365-369. McMillen, J., M. S. Center, and R. A. Consigli. 1976. Origin of the polyoma virus-associated endonuclease. J. Virol. 17:127-131. Monjardino, J., and A. W. James. 1975. Denaturation of polyoma DNA by phage T4 gene 32 protein. Nature (London) 255:249-252. Morrow, J. F., and P. Berg. 1972. Cleavage of simian virus 40 DNA at a unique site by a bacterial restriction enzyme. Proc. Natl. Acad. Sci. U.S.A. 69:33653369. Parodi, A., P. Rouget, 0. Croissant, D. Blangy, and F. Cuzin. 1974. Endonucleolytic cleavage of polyoma virus DNA: general properties and site specificity of the virion-associated endonuclease. Cold Spring Harbor Symp. Quant. Biol. 39:247-254. Vogt, M., and R. Dulbecco. 1962. Studies on cells rendered neoplastic by polyoma virus. Virology 16:41-51. Winocour, E. 1963. Purification of polyoma virus. Virology 19:158-168. Yaniv, M., A. Chestier, C. Dauguet, and 0. Croissant. 1975. Location of the T4 gene 32 protein binding site on polyoma virus DNA. FEBS Lett. 57:126-129. Yaniv, M., 0. Croissant, and F. Cuzin. 1973. Location of the T4 gene 32 protein binding sites on polyoma virus DNA. Biochem. Biophys. Res. Commun. 57:1074-1079.