VIROLOGY
99,
152-157 (1979)
Methylation U. v. ACKEN, Robert
of Viral DNA in Vivo and in Vitro
D. SIMON,’
Koch-Institut,
Abteilung
F. GRUNERT, Biochemie, Accepted
H.-P.
Nordufer July
20,
DORING, D-1000 Berlin
AND H. KRiiGER 65, Germany
26, 1979
Methylation of herpesvirus saimiri DNA and adenovirus type 5 DNA was examined by labelling with [6-3H]uridine and [VH]adenosine and separating the bases by thin layer chromatography. The number of methyl groups was less than one per genome in both DNAs, indicating that these viral DNAs are not substrates for methylation in tivo. In a long-term in vitro incubation with purified rat liver DNA methylase adenovirus type 5 DNA and herpes simplex virus type I DNA were methylated to about the same degree as M. luteus DNA (1 methyl group per 42 bases). In a short-term incubation the viral DNAs accepted considerably less methyl groups than M. luteus DNA. This suggests that viral DNAs have lower affinity for DNA methylases than bacterial DNAs. Rat liver DNA was a competitive inhibitor for the in vitro methylation of adenovirus type 5 DNA by rat liver DNA methylase. The higher affinity of the enzyme for cellular DNA may explain why viral DNA is not methylated in the cell.
example, the only methyl group of phage 4x 174 seems to be essential for the deThe role of DNA methylation in pro- velopment of this phage, probably as a karyotes as part of the modification-rerecognition signal for a specific endonustriction system has been described else- clease (Friedman et aZ., 1977). In phage where (for reviews see Meselson et al., fd the modification methylation comprises 19’72; Arber, 1974). But only a small part of only four methyl groups (see Meselson et highly specific methylation is involved in al., 1972). this phenomenon: The function of the We therefore thought it interesting to greater part of DNA methylation in bac- examine the methylation of some more teria as well as the function of DNA methylDNA viruses with sensitive methods. Our ation in eucaryotes is still unknown. results with herpesvirus saimiri and adenoDNA viruses which can be grown on virus type 5 confirm that in general the mammalian cells, would be expected to DNA of DNA viruses is not methylated in serve as substrates for eucaryotic DNA viva. To find out the reason for this phemethylases. However, the DNA of the nomenon we have studied the in vitro small animal virus polyoma and the DNA methylation of some viral DNAs with parof the large human virus herpes simplex tially purified DNA methylases from rat virus type I have been reported to be liver and Hela cells. unmethylated (Kaye and Winocour, 1967; Low et al., 1969). On the other hand, small MATERIALS AND METHODS amounts of 5’-methylcytosine and iVRadiochemicals and scintillation media. methyladenine have recently been found in the DNA of adenovirus types 2 and 12 S-Adenosyl-L-[methyZ-3H]methionine (SAM) (5-10.5 Ci/mmol, 1 mCi/ml), [2(Gtinthert et al., 1976). (20-25 Ci/mmol), [6-3H]There are some cases where even a very 3H]adenosine uridine (5-15 Ci/mmol, 1 mCi/ml), and small number of methyl groups on a DNA can be of considerable importance. For desoxy [14C]cytidine (405-540 mCi/mmol) were obtained from Amersham. The la1 To whom reprint requests should be addressed. belled SAM was diluted to 2.2 Ci/mmol, INTRODUCTION
0042~6822/79/150152-06$02.00/0 Copyright All rights
8 1979 by Academic Press, Inc. of reproduction in any form reserved.
152
METHYLATION
0.2 mCi/ml with unlabelled S-adenosyl-Lmethionine from Boehringer-Mannheim. [53H]Methylcytosine was prepared in the following way: Escherichia coli B soluble RNA was methylated with S-adenosyl-L[methyZ-3H]methionine with a crude extract of rat liver transfer RNA methylases (Kahle et al., 1971). The RNA was extracted, hydrolysed, and [5-3H]methylcytosine isolated by chromatography on Dowex (Craddock, 1969). A toluene or toluene-Triton X-100 (3:l) scintillation medium was used. Toluene scintillation medium contained 0.01% POPOP and 0.4% PPO. Enzymes and chemicals. RNase A and Pronase were from Boehringer-Mannheim. 5’-Methylcytosine and N6-methyladenine were from Calbiochem, DEAE Sephadex A50 from Pharmacia, Durrum Al from the Durrum Chemical Corporation (California). M. Zuteus DNA was from Miles and salmon sperm DNA from Serva. Growth of virus and purifiation of viral DNAs. Herpesvirus saimiri, which was a
gift from Dr. B. Fleckenstein, Erlangen, was grown on owl monkey kidney (OMK) cells in the presence of 2 j&i [6-3H]uridine/ ml (5 Ci/mmol). Virus and viral DNA was purified on sucrose gradients as described by Fleckenstein and Wolf (1974). Adenovirus type 5 was grown on HeLa cells in the presence of 2 @Zi [6-3H]uridine/ ml (5 Ci/mmol) or 1 &i [2-3H]adenosine/ ml (10 Ci/mmol). The virus was isolated on a CsCl gradient (Green and Pina, 1963). The viral DNA was purified on a sucrose gradient as described for herpesvirus saimiri, the only difference being that centrifugation in the SW 27 rotor was for 13 hr at 27,000 rpm. The purity of the viral DNAs was controlled by CsCl density gradient centrifugation in an analytical ultracentrifuge. Thin layer chromatography. The labelled viral DNA together with 50 pg salmon sperm DNA was incubated in 0.5 M NaOH for 2 hr at 37”, precipitated by TCA, and hydrolysed in 88% formic acid at 175” for 40 min. After drying and adding 2.5 pg 5’methylcytosine, the hydrolysate was subjected to two-dimensional cellulose thin layer chromatography with 130 ml iso-
OF
VIRAL
DNA
153
propanol, 34 ml HCl (37%), and 36 ml H,O in the first dimension and 174 ml butanol, 36 ml 0.2 M ammonia in the second dimension. Cytosine and 5’-methylcytosine spots were scraped off and after the addition of 4 pg 5’-methylcytosine to the 5’-methylcytosine containing cellulose both were eluted with 0.5 ml of 0.2 M HCl. The eluate was rechromatographed in the two dimensions described above. The cytosine and 5’methylcytosine containing spots were extracted and the radioactivity counted (Kahle et al., 1971). For the separation of adenine and N6methyladenine the same chromatographic procedure was used, but they were eluted with 0.5 M HCl and counted in a Triton XlOO-toluene scintillation mixture. Base analysis of cellular DNA. DNA from nuclei of OMK cells and HeLa cells was hydrolysed and the bases separated on a Durrum DC 1A column (Baur et al., from this laboratory, in preparation). Extraction of DNA methylases. DNA methylases were extracted from purified nuclei of regenerating rat liver or HeLa cells by high salt and purified by step elution from DEAE Sephadex A50 (Simon et al., 1978). Methylation assay. The assay mixture for the rat liver methylase with denatured DNA contained in a total volume of 0.2 ml: 10 n&I Tris-HCl buffer, pH 7.8, 5 mM EDTA, 0.1 mM dithiothreithol (DTT), 0.01% Triton X-100, 150 mM KCl, 4.5 $I! SAM (=2 PCi), denatured DNA (10 min 100°), and the enzyme. Native DNA was methylated in lower salt concentration (50 m&f KCl). Short-term methylation was performed for 30 min at 37”, long-term methylation for 36 hr at 33” (Simon et al., 1978). The incubation mixture for the HeLa cell DNA methylase was as described by Roy and Weissbach (1975). After the incubation the DNA was extracted with chloroformisoamyl alcohol, treated with NaOH, precipitated on GFC filters, and counted (Simon et al., 1978). RESULTS
To measure the amount of 5’-methylcytosine the viral DNA was labelled in cyto-
154
VAN ACKEN
sine and 5’-methylcytosine by growing virus in the presence of [6-3H]uridine. Purified viral DNA was hydrolysed in formic acid and cytosine and 5’-methylcytosine were separated by chromatography and rechromatography on cellulose thin layer. The number of methyl groups per viral genome was calculated by comparing the radioactivity in 5’-methylcytosine with that in cytosine. The chromatography system, starting with the formic acid hydrolysis and including the elution of the bases after the first chromatography and rechromatography, was initially checked with [14C]cytosine prepared from [UJ4C]deoxycytidine and [5-3H]methylcytosine prepared as described under Materials and Methods. The results, showing the same recovery for cytosine and for 5’-methylcytosine and a good reproducibility, proved the reliability of the method. When labelled DNA from herpesvirus saimiri (total cpm 1.5 x 106) was analysed, 501,000 cpm was found in cytosine and 7.5 cpm in 5’-methylcytosine (Table 1). Based on a molecular weight of 83 x lo6 and a G + C content of 45%, one genome contains about 56,500 cytosines. With one 5’-methylcytosine per viral genome we should expect 1 cpm in 5’-methylcytosine per 56,500 cpm in cytosine and consequently 8.8 cpm in 5’methylcytosine per 501,000 cpm in cytosine. As we only found 7.5 cpm in 5’-methylcytosine, the herpesvirus saimiri genome contains less than one 5’-methylcytosine. Adenovirus type 5 DNA containing 4.5 x lo6 cpm from the [6-3H]uridine label was treated in the same way. With a molecular weight of 25 x lo6 and a G + C content of 58%, adenovirus DNA contains about 22,040 cytosines per genome. After chromatography of the DNA 1.747 x lo6 cpm was found in cytosine and 77 cpm in 5’-methylcytosine. This is equivalent to only 0.44 5’methylcytosines per genome. Since Gtinthert et al. (1976) reported a small number of N6-methyladenines in adenovirus types 2s and 12 DNA, adenovirus type 5 was also grown in the presence of [2-3H]adenosine and the DNA extracted (see Materials and Methods). The DNA was hydrolysed and chromatographed twice as
ET AL. TABLE BASE ANALYSIS
DNA Adenovirus 5 Herpesvirus saimiri
1
OF LABELED
VIRAL
DNA”
Cytosine kpm)
F-Methylcytosine (cpm)
CH,/viral genome
1,747,ooo
77
0.44
501,000
7.5
0.85
n Adenovirus type 5 and herpesvirus saimiri were grown in the presence of [3H]uridine. The viral DNA was purified and the bases were separated by thin layer chromatography and rechromatography as detailed under Materials and Methods.
described above, except that N6-methyladenine was added instead of 5’-methylcytosine. Adenovirus type 5 DNA contains about 15,910 adenines. We found 289,500 cpm in adenine and 12 cpm in N6-methyladenine, equivalent to 0.67 N6-methyladenines per genome. In contrast to viral DNAs, host cell DNA showed a normal methylation extent. The amount of 5’-methylcytosine determined by base analysis on Dumum Al was 0.6% for all bases for HeLa cell DNA and 0.8% for OMK cell DNA. N6-Methyladenine was not found. In Vitro Methylation
of Viral DNA
To throw some light on how viral DNA escapes methylation in viva, we studied its in vitro methylation with partially purified DNA methylases from rat liver and from HeLa cells. We mostly used the enzyme from rat liver because it is relatively easy to purify in the required amounts. Moreover this enzyme has been shown to have the same sequence specificity in vitro as in vivo (unpublished results). The methylation sequence is dCpdG for all eukaryotic DNA methylases described so far (see Discussion). As viral substrate DNA we mainly used adenovirus type 5 DNA, and in some cases herpes simplex virus type I. Table 2 shows the extent of methylation of the two viral DNAs and 1M. Zuteus DNA in a short-term and a long-term incubation with rat liver DNA methylase. In the long-
METHYLATION
OF VIRAL
TABLE
DNA
155
2
SHORT-TERM AND LONG-TERMMETHYLATIONOFVIRALDNA INVITRO~ CH, incorporation Substrate
30 min at 37 kpm)
CHJlOO bases
(cpm)
CHJlOO bases
luteus DNA Ad5 DNA HSV I DNA
9,970 5,140 5,800
0.89 0.46 0.52
26,950 22,530
2.4 2.0 2.4
M.
36 hr at 33
26,950
a Native adenovirus type 5 DNA (Ad5), 0.38 pg, herpes simplex virus type I (HSV I), or M. luteus DNA was incubated with rat liver DNA methylase in the assay for double-stranded DNA for 30 min at 37” or 36 hr at 33”. For details see Materials and Methods.
term incubation, which under certain conditions is a measure of the number of substrate sites, herpesvirus and adenovirus DNA were methylated to about the same extent as M. luteus DNA. M. luteus DNA is the best natural substrate known for the DNA methylase from rat liver and in this incubation accepted 1 methyl group per 42 bases. In the short-term incubation with rat liver methylase, however, both viral DNAs accepted only half the amount of methyl groups compared with M. luteus DNA. This difference in methylation between viral DNA and M. Zuteus DNA in the shortterm incubation became more obvious when HeLa cell DNA methylase was used for methylation (Table 3). While the rat liver DNA methylase again methylated adenovirus type 5 DNA to one-half the extent
compared with M. luteus DNA, the HeLa cell DNA methylase methylated adenovirus type 5 DNA only to the extent of onefifth. To exclude the possibility of the methylase preparation from HeLa cells containing a factor which binds preferentially to viral DNA and inhibits its methylation, the combined enzyme preparations from HeLa cells and rat liver were incubated with adenovirus type 5 DNA. There was no inhibition of the rat liver DNA methylase in the combined assay. The difference in the extent of methylation in the short-term incubation under limiting DNA concentration suggested a lower binding affinity of the DNA methylases for viral DNAs. We therefore considered whether nuclear DNA, although almost completely methylTABLE 3 ated in vivo and therefore only a very poor substrate in vitro, might effectively ACTIVITY OFTHEDNA METHYLASES FROMRAT LIVERANDHeLa CELLSWITHADENOVIRUSTYPE 5 compete with viral DNA for the DNA methylase. As DNA methylases remain DNACOMPAREDTO M.luteus DNA” bound to chromatin upon the isolation of nuclei at low ionic strength, a certain afSource of DNA cpm in cpm in methylase Ad5 DNA M. luteus DNA finity to methylated DNA seemed quite probable. HeLa cells 765 4125 For competition experiments with viral Rat liver 1950 4350 DNA and cellular DNA in the same assay, Both, HeLa cells native nuclear DNA from rat liver, which and rat liver 2638 still accepts about 1 methyl group per 600 bases, was methylated to saturation with a DNA methylases, either separately or combined, purified rat liver methylase in vitro using were incubated with 0.5 pg of adenovirus type 5 DNA unlabelled SAM (see Materials and Meth(Ad5) or 0.5 pg of M. lute-m DNA in the standard assay for the HeLa cell enzyme at pH 6.5 (see Ma- ods). This was done to avoid a background terials and Methods). methylation of rat liver DNA in experi-
156
VAN
ACKEN
ments where viral DNA and cellular DNA were incubated together. The same results, however, were obtained when this step was omitted. Rat liver DNA methylase was incubated with adenovirus type 5 DNA and increasing amounts of exhaustively methylated rat liver DNA, in the standard assay for doublestranded DNA. As shown in Table 4, there was a considerable competitive effect of nuclear DNA on the methylation of viral DNA. DISCUSSION
Our results confirm that viral DNAs are not methylated in viva or that at least no methylated viral DNA appears in infectious virus particles. The few methyl groups in 5’-methylcytosine as well as in P-methyladenine of adenovirus types 2 and 12 DNA reported by Gunther% et al. (19’76) could be either due to strain differences, or to contamination with cellular DNA, or to an artifact in the thin layer chromatography. In contrast to our chromatography system, in their rechromatography system the methylated bases chromatographed behind the corresponding labelled unmethylated bases. There are two explanations as to why methylation of viral DNA does not take place in viva: first, DNA methylation could somehow be shut off upon infection. This possibility has been excluded by the experiments of Gunther? et al. (1976) and Sharma and Biswal (1977). Second, viral DNAs could be poor substrates for the DNA methylases, either due to lack of substrate sequences or to lack of binding sequences for the enzymes. It has been shown that the binding sequences of the DNA methylase from rat liver are not identical with the substrate sequences (Drahovsky and Morris, 1972). The main and possibly the only substrate sequence methylated by the eucaryotic DNA methylases described so far is dCpdG (Roy and Weissbach, 1975; Grippo et al., 1968). dCpdG is contained in about fivefold reduced amounts in the DNA of small animal viruses, but in random amounts in the DNA of large animal viruses as herpes simplex virus type I (Russel et al., 1977; SubakSharpe et al., 1966).
ET AL. TABLE
4
METHYLATION OF ADENOVIRUS TYPE 5 DNA WITH RAT LIVER DNA METHYLASE IN THE PRESENCE OF RAT LIVER DNA” Ratio Ad5 DNA: cpm in Ad5 DNA
cell DNA
I:0 1:l 1:2 1:5
32,200
13,830 6,869 2,160
“ Rat liver DNA methylase was incubated with 1 pg of adenovirus type 5 DNA (Ad5) and increasing amounts of nuclear rat liver DNA in the standard assay for double-stranded DNA for 30 min at 37”. Rat liver DNA had been exhaustively methylated in vitro with unlabelled SAM previously.
The results of our long-term methylation likewise showed that there is no lack of substrate sites in herpesvirus and adenovirus DNA. In contrast to a long-term incubation, in a short-term incubation both viral DNAs accepted only one-half the amount of methyl groups compared with M. luteus DNA using the rat liver DNA methylase and onefifth the amount using the HeLa cell DNA methylase. This could be best explained by a lower binding affinity of eucaryotic methylases for viral DNA. Our competition experiments with viral DNA and cellular DNA in the same assay confirmed a lower affinity of the rat liver DNA methylase for viral DNA compared with cellular DNA and thus offer an explanation of how methylation of viral DNA could be prevented in the cell. ACKNOWLEDGMENTS
We thank M. Klewer and P. Hansen for excellent technical assistence. This work has been supported by the Deutsche Forschungsgemeinschaft. REFERENCES
ARBER, W. (1974). Restriction and modification of DNA. Prop-. Nucleic Acids Res. 14, l-37. CRADDOCK, V. M. (1969). Methylation of transfer RNA and of ribosomal RNA in rat liver in the intact animal and the effect of carcinogens. Biochim. Btiphys. Acta 195, 351-369. DRAHOVSKY, D., and MORRIS, N. R. (1972). The mechanism of action of rat liver DNA methylase.
METHYLATION Nucleotide requirements for binding and methylation. B&him. Biophys. Acta 277, 245-250. FLECKENSTEIN, B., and WOLF, H. (1974). Purification and properties of herpesvirus saimiri DNA. Virology
58, 55-64.
FRIEDMAN, J., FRIEDMAN, A., and RAZIN, A. (1977). Studies on the biological role of DNA methylation in excision of one-genome long single-stranded 4x 174 DNA. Nucleic Acids Res. 4, 3483-3496. GREEN, M., and PIRA, M. (1963). Biochemical studies on adenovirus multiplication. Vimlogy 20, 199-207. GRIPPO, P., JACCARINO, M., PARISI, E., and SCARANO, E. (1968). Methylation of DNA in developing sea urchin embryos. J. Mol. Biol. 36, 195-208. G~NTHERT, U., SCHWEIGER, M., STUPP, M., and DOERFLER, W. (1976). DNA methylation in adenovirus, adenovirus-transformed cells and host cells. hoc. Nut. Acad. Sci USA 73(11), 3923-3927. KAHLE, P., HOPPE-SEYLER, P., and KRUGER, H. (1971). Transfer RNA methylases from rat liver nuclei. B&him. Biophys. Acta 240, 384-391. KAYE, A. M., and WINOCOUR, E. (1967). On the 5methylcytosine found in the DNA extracted from polyoma virus. J. Mol. Biol. 24, 475-478. Low, M., HAY, J., and KEIR, H. (1969). DNA of herpes simplex virus is not a substrate for methylation in vivo. J. Mol. Biol. 46, 205-207. MESELSON, M., YUAN, R., and HEYWOOD, J. (1972). Restriction and modification of DNA. Annu. Rev. Biochem. 41, 447-466.
OF VIRAL
DNA
157
ROY, P. H., and WEISSBACH, A. (1975). DNA methylaae from HeLa cell nuclei. Nucleic Acids Res. 2, 1669- 1684. RUSSEL, G. J., WALKER, P. M. B., ELTON, R. A., and SUBAK-SHARPE, J. H. (1976). Doublet frequency analysis of fractionated vertebrate nuclear DNA. J. Mol. Biol. 108, l-23. SHARMA, S., and BISWAL, N. (1977). Studies on the in vivo methylation of replicating herpes simplex virus type I DNA. ViTology 82, 265-274. SIMON, D., GRUNERT, F., VAN ACKEN, U., DORING, H. P., and KRUGER, H. (1978). DNA methylase from regenerating rat liver: Purification and characterisation. Nucleic Acids Res. 5, 2153-2167. SNEIDER, T. W., TEAGUE, W. M., and ROGACHEVSKY, L. M. (1975). S-Adenosylmethionine: DNAcytosine 5-methyltransferase from a Novikoff rat hepatoma cell line. Nucleic Acids Res. 2,1685-1700. SUBAK-SHARPE, H., BORK, R. R., CRAWFORD, L. V., MORRISON, J. M., HAY, J., and KEIR, H. M. (1966). An approach to the evolutionary relationship of mammalian DNA viruses through analysis of the pattern of nearest neighbour base sequences. Cold Spring Harbor Symp. Qwxnt. Biol. 31, 737-748. TURNBULL, J. F., and ADAMS, R. L. P. (1976). DNA methylase: Purification from ascites cells and effect of various DNA substrates on its activity. Nucleic Acids
Res.
3, 677-695.
99,
152-157 (1979)
Methylation U. v. ACKEN, Robert
of Viral DNA in Vivo and in Vitro
D. SIMON,’
Koch-Institut,
Abteilung
F. GRUNERT, Biochemie, Accepted
H.-P.
Nordufer July
20,
DORING, D-1000 Berlin
AND H. KRiiGER 65, Germany
26, 1979
Methylation of herpesvirus saimiri DNA and adenovirus type 5 DNA was examined by labelling with [6-3H]uridine and [VH]adenosine and separating the bases by thin layer chromatography. The number of methyl groups was less than one per genome in both DNAs, indicating that these viral DNAs are not substrates for methylation in tivo. In a long-term in vitro incubation with purified rat liver DNA methylase adenovirus type 5 DNA and herpes simplex virus type I DNA were methylated to about the same degree as M. luteus DNA (1 methyl group per 42 bases). In a short-term incubation the viral DNAs accepted considerably less methyl groups than M. luteus DNA. This suggests that viral DNAs have lower affinity for DNA methylases than bacterial DNAs. Rat liver DNA was a competitive inhibitor for the in vitro methylation of adenovirus type 5 DNA by rat liver DNA methylase. The higher affinity of the enzyme for cellular DNA may explain why viral DNA is not methylated in the cell.
example, the only methyl group of phage 4x 174 seems to be essential for the deThe role of DNA methylation in pro- velopment of this phage, probably as a karyotes as part of the modification-rerecognition signal for a specific endonustriction system has been described else- clease (Friedman et aZ., 1977). In phage where (for reviews see Meselson et al., fd the modification methylation comprises 19’72; Arber, 1974). But only a small part of only four methyl groups (see Meselson et highly specific methylation is involved in al., 1972). this phenomenon: The function of the We therefore thought it interesting to greater part of DNA methylation in bac- examine the methylation of some more teria as well as the function of DNA methylDNA viruses with sensitive methods. Our ation in eucaryotes is still unknown. results with herpesvirus saimiri and adenoDNA viruses which can be grown on virus type 5 confirm that in general the mammalian cells, would be expected to DNA of DNA viruses is not methylated in serve as substrates for eucaryotic DNA viva. To find out the reason for this phemethylases. However, the DNA of the nomenon we have studied the in vitro small animal virus polyoma and the DNA methylation of some viral DNAs with parof the large human virus herpes simplex tially purified DNA methylases from rat virus type I have been reported to be liver and Hela cells. unmethylated (Kaye and Winocour, 1967; Low et al., 1969). On the other hand, small MATERIALS AND METHODS amounts of 5’-methylcytosine and iVRadiochemicals and scintillation media. methyladenine have recently been found in the DNA of adenovirus types 2 and 12 S-Adenosyl-L-[methyZ-3H]methionine (SAM) (5-10.5 Ci/mmol, 1 mCi/ml), [2(Gtinthert et al., 1976). (20-25 Ci/mmol), [6-3H]There are some cases where even a very 3H]adenosine uridine (5-15 Ci/mmol, 1 mCi/ml), and small number of methyl groups on a DNA can be of considerable importance. For desoxy [14C]cytidine (405-540 mCi/mmol) were obtained from Amersham. The la1 To whom reprint requests should be addressed. belled SAM was diluted to 2.2 Ci/mmol, INTRODUCTION
0042~6822/79/150152-06$02.00/0 Copyright All rights
8 1979 by Academic Press, Inc. of reproduction in any form reserved.
152
METHYLATION
0.2 mCi/ml with unlabelled S-adenosyl-Lmethionine from Boehringer-Mannheim. [53H]Methylcytosine was prepared in the following way: Escherichia coli B soluble RNA was methylated with S-adenosyl-L[methyZ-3H]methionine with a crude extract of rat liver transfer RNA methylases (Kahle et al., 1971). The RNA was extracted, hydrolysed, and [5-3H]methylcytosine isolated by chromatography on Dowex (Craddock, 1969). A toluene or toluene-Triton X-100 (3:l) scintillation medium was used. Toluene scintillation medium contained 0.01% POPOP and 0.4% PPO. Enzymes and chemicals. RNase A and Pronase were from Boehringer-Mannheim. 5’-Methylcytosine and N6-methyladenine were from Calbiochem, DEAE Sephadex A50 from Pharmacia, Durrum Al from the Durrum Chemical Corporation (California). M. Zuteus DNA was from Miles and salmon sperm DNA from Serva. Growth of virus and purifiation of viral DNAs. Herpesvirus saimiri, which was a
gift from Dr. B. Fleckenstein, Erlangen, was grown on owl monkey kidney (OMK) cells in the presence of 2 j&i [6-3H]uridine/ ml (5 Ci/mmol). Virus and viral DNA was purified on sucrose gradients as described by Fleckenstein and Wolf (1974). Adenovirus type 5 was grown on HeLa cells in the presence of 2 @Zi [6-3H]uridine/ ml (5 Ci/mmol) or 1 &i [2-3H]adenosine/ ml (10 Ci/mmol). The virus was isolated on a CsCl gradient (Green and Pina, 1963). The viral DNA was purified on a sucrose gradient as described for herpesvirus saimiri, the only difference being that centrifugation in the SW 27 rotor was for 13 hr at 27,000 rpm. The purity of the viral DNAs was controlled by CsCl density gradient centrifugation in an analytical ultracentrifuge. Thin layer chromatography. The labelled viral DNA together with 50 pg salmon sperm DNA was incubated in 0.5 M NaOH for 2 hr at 37”, precipitated by TCA, and hydrolysed in 88% formic acid at 175” for 40 min. After drying and adding 2.5 pg 5’methylcytosine, the hydrolysate was subjected to two-dimensional cellulose thin layer chromatography with 130 ml iso-
OF
VIRAL
DNA
153
propanol, 34 ml HCl (37%), and 36 ml H,O in the first dimension and 174 ml butanol, 36 ml 0.2 M ammonia in the second dimension. Cytosine and 5’-methylcytosine spots were scraped off and after the addition of 4 pg 5’-methylcytosine to the 5’-methylcytosine containing cellulose both were eluted with 0.5 ml of 0.2 M HCl. The eluate was rechromatographed in the two dimensions described above. The cytosine and 5’methylcytosine containing spots were extracted and the radioactivity counted (Kahle et al., 1971). For the separation of adenine and N6methyladenine the same chromatographic procedure was used, but they were eluted with 0.5 M HCl and counted in a Triton XlOO-toluene scintillation mixture. Base analysis of cellular DNA. DNA from nuclei of OMK cells and HeLa cells was hydrolysed and the bases separated on a Durrum DC 1A column (Baur et al., from this laboratory, in preparation). Extraction of DNA methylases. DNA methylases were extracted from purified nuclei of regenerating rat liver or HeLa cells by high salt and purified by step elution from DEAE Sephadex A50 (Simon et al., 1978). Methylation assay. The assay mixture for the rat liver methylase with denatured DNA contained in a total volume of 0.2 ml: 10 n&I Tris-HCl buffer, pH 7.8, 5 mM EDTA, 0.1 mM dithiothreithol (DTT), 0.01% Triton X-100, 150 mM KCl, 4.5 $I! SAM (=2 PCi), denatured DNA (10 min 100°), and the enzyme. Native DNA was methylated in lower salt concentration (50 m&f KCl). Short-term methylation was performed for 30 min at 37”, long-term methylation for 36 hr at 33” (Simon et al., 1978). The incubation mixture for the HeLa cell DNA methylase was as described by Roy and Weissbach (1975). After the incubation the DNA was extracted with chloroformisoamyl alcohol, treated with NaOH, precipitated on GFC filters, and counted (Simon et al., 1978). RESULTS
To measure the amount of 5’-methylcytosine the viral DNA was labelled in cyto-
154
VAN ACKEN
sine and 5’-methylcytosine by growing virus in the presence of [6-3H]uridine. Purified viral DNA was hydrolysed in formic acid and cytosine and 5’-methylcytosine were separated by chromatography and rechromatography on cellulose thin layer. The number of methyl groups per viral genome was calculated by comparing the radioactivity in 5’-methylcytosine with that in cytosine. The chromatography system, starting with the formic acid hydrolysis and including the elution of the bases after the first chromatography and rechromatography, was initially checked with [14C]cytosine prepared from [UJ4C]deoxycytidine and [5-3H]methylcytosine prepared as described under Materials and Methods. The results, showing the same recovery for cytosine and for 5’-methylcytosine and a good reproducibility, proved the reliability of the method. When labelled DNA from herpesvirus saimiri (total cpm 1.5 x 106) was analysed, 501,000 cpm was found in cytosine and 7.5 cpm in 5’-methylcytosine (Table 1). Based on a molecular weight of 83 x lo6 and a G + C content of 45%, one genome contains about 56,500 cytosines. With one 5’-methylcytosine per viral genome we should expect 1 cpm in 5’-methylcytosine per 56,500 cpm in cytosine and consequently 8.8 cpm in 5’methylcytosine per 501,000 cpm in cytosine. As we only found 7.5 cpm in 5’-methylcytosine, the herpesvirus saimiri genome contains less than one 5’-methylcytosine. Adenovirus type 5 DNA containing 4.5 x lo6 cpm from the [6-3H]uridine label was treated in the same way. With a molecular weight of 25 x lo6 and a G + C content of 58%, adenovirus DNA contains about 22,040 cytosines per genome. After chromatography of the DNA 1.747 x lo6 cpm was found in cytosine and 77 cpm in 5’-methylcytosine. This is equivalent to only 0.44 5’methylcytosines per genome. Since Gtinthert et al. (1976) reported a small number of N6-methyladenines in adenovirus types 2s and 12 DNA, adenovirus type 5 was also grown in the presence of [2-3H]adenosine and the DNA extracted (see Materials and Methods). The DNA was hydrolysed and chromatographed twice as
ET AL. TABLE BASE ANALYSIS
DNA Adenovirus 5 Herpesvirus saimiri
1
OF LABELED
VIRAL
DNA”
Cytosine kpm)
F-Methylcytosine (cpm)
CH,/viral genome
1,747,ooo
77
0.44
501,000
7.5
0.85
n Adenovirus type 5 and herpesvirus saimiri were grown in the presence of [3H]uridine. The viral DNA was purified and the bases were separated by thin layer chromatography and rechromatography as detailed under Materials and Methods.
described above, except that N6-methyladenine was added instead of 5’-methylcytosine. Adenovirus type 5 DNA contains about 15,910 adenines. We found 289,500 cpm in adenine and 12 cpm in N6-methyladenine, equivalent to 0.67 N6-methyladenines per genome. In contrast to viral DNAs, host cell DNA showed a normal methylation extent. The amount of 5’-methylcytosine determined by base analysis on Dumum Al was 0.6% for all bases for HeLa cell DNA and 0.8% for OMK cell DNA. N6-Methyladenine was not found. In Vitro Methylation
of Viral DNA
To throw some light on how viral DNA escapes methylation in viva, we studied its in vitro methylation with partially purified DNA methylases from rat liver and from HeLa cells. We mostly used the enzyme from rat liver because it is relatively easy to purify in the required amounts. Moreover this enzyme has been shown to have the same sequence specificity in vitro as in vivo (unpublished results). The methylation sequence is dCpdG for all eukaryotic DNA methylases described so far (see Discussion). As viral substrate DNA we mainly used adenovirus type 5 DNA, and in some cases herpes simplex virus type I. Table 2 shows the extent of methylation of the two viral DNAs and 1M. Zuteus DNA in a short-term and a long-term incubation with rat liver DNA methylase. In the long-
METHYLATION
OF VIRAL
TABLE
DNA
155
2
SHORT-TERM AND LONG-TERMMETHYLATIONOFVIRALDNA INVITRO~ CH, incorporation Substrate
30 min at 37 kpm)
CHJlOO bases
(cpm)
CHJlOO bases
luteus DNA Ad5 DNA HSV I DNA
9,970 5,140 5,800
0.89 0.46 0.52
26,950 22,530
2.4 2.0 2.4
M.
36 hr at 33
26,950
a Native adenovirus type 5 DNA (Ad5), 0.38 pg, herpes simplex virus type I (HSV I), or M. luteus DNA was incubated with rat liver DNA methylase in the assay for double-stranded DNA for 30 min at 37” or 36 hr at 33”. For details see Materials and Methods.
term incubation, which under certain conditions is a measure of the number of substrate sites, herpesvirus and adenovirus DNA were methylated to about the same extent as M. luteus DNA. M. luteus DNA is the best natural substrate known for the DNA methylase from rat liver and in this incubation accepted 1 methyl group per 42 bases. In the short-term incubation with rat liver methylase, however, both viral DNAs accepted only half the amount of methyl groups compared with M. luteus DNA. This difference in methylation between viral DNA and M. Zuteus DNA in the shortterm incubation became more obvious when HeLa cell DNA methylase was used for methylation (Table 3). While the rat liver DNA methylase again methylated adenovirus type 5 DNA to one-half the extent
compared with M. luteus DNA, the HeLa cell DNA methylase methylated adenovirus type 5 DNA only to the extent of onefifth. To exclude the possibility of the methylase preparation from HeLa cells containing a factor which binds preferentially to viral DNA and inhibits its methylation, the combined enzyme preparations from HeLa cells and rat liver were incubated with adenovirus type 5 DNA. There was no inhibition of the rat liver DNA methylase in the combined assay. The difference in the extent of methylation in the short-term incubation under limiting DNA concentration suggested a lower binding affinity of the DNA methylases for viral DNAs. We therefore considered whether nuclear DNA, although almost completely methylTABLE 3 ated in vivo and therefore only a very poor substrate in vitro, might effectively ACTIVITY OFTHEDNA METHYLASES FROMRAT LIVERANDHeLa CELLSWITHADENOVIRUSTYPE 5 compete with viral DNA for the DNA methylase. As DNA methylases remain DNACOMPAREDTO M.luteus DNA” bound to chromatin upon the isolation of nuclei at low ionic strength, a certain afSource of DNA cpm in cpm in methylase Ad5 DNA M. luteus DNA finity to methylated DNA seemed quite probable. HeLa cells 765 4125 For competition experiments with viral Rat liver 1950 4350 DNA and cellular DNA in the same assay, Both, HeLa cells native nuclear DNA from rat liver, which and rat liver 2638 still accepts about 1 methyl group per 600 bases, was methylated to saturation with a DNA methylases, either separately or combined, purified rat liver methylase in vitro using were incubated with 0.5 pg of adenovirus type 5 DNA unlabelled SAM (see Materials and Meth(Ad5) or 0.5 pg of M. lute-m DNA in the standard assay for the HeLa cell enzyme at pH 6.5 (see Ma- ods). This was done to avoid a background terials and Methods). methylation of rat liver DNA in experi-
156
VAN
ACKEN
ments where viral DNA and cellular DNA were incubated together. The same results, however, were obtained when this step was omitted. Rat liver DNA methylase was incubated with adenovirus type 5 DNA and increasing amounts of exhaustively methylated rat liver DNA, in the standard assay for doublestranded DNA. As shown in Table 4, there was a considerable competitive effect of nuclear DNA on the methylation of viral DNA. DISCUSSION
Our results confirm that viral DNAs are not methylated in viva or that at least no methylated viral DNA appears in infectious virus particles. The few methyl groups in 5’-methylcytosine as well as in P-methyladenine of adenovirus types 2 and 12 DNA reported by Gunther% et al. (19’76) could be either due to strain differences, or to contamination with cellular DNA, or to an artifact in the thin layer chromatography. In contrast to our chromatography system, in their rechromatography system the methylated bases chromatographed behind the corresponding labelled unmethylated bases. There are two explanations as to why methylation of viral DNA does not take place in viva: first, DNA methylation could somehow be shut off upon infection. This possibility has been excluded by the experiments of Gunther? et al. (1976) and Sharma and Biswal (1977). Second, viral DNAs could be poor substrates for the DNA methylases, either due to lack of substrate sequences or to lack of binding sequences for the enzymes. It has been shown that the binding sequences of the DNA methylase from rat liver are not identical with the substrate sequences (Drahovsky and Morris, 1972). The main and possibly the only substrate sequence methylated by the eucaryotic DNA methylases described so far is dCpdG (Roy and Weissbach, 1975; Grippo et al., 1968). dCpdG is contained in about fivefold reduced amounts in the DNA of small animal viruses, but in random amounts in the DNA of large animal viruses as herpes simplex virus type I (Russel et al., 1977; SubakSharpe et al., 1966).
ET AL. TABLE
4
METHYLATION OF ADENOVIRUS TYPE 5 DNA WITH RAT LIVER DNA METHYLASE IN THE PRESENCE OF RAT LIVER DNA” Ratio Ad5 DNA: cpm in Ad5 DNA
cell DNA
I:0 1:l 1:2 1:5
32,200
13,830 6,869 2,160
“ Rat liver DNA methylase was incubated with 1 pg of adenovirus type 5 DNA (Ad5) and increasing amounts of nuclear rat liver DNA in the standard assay for double-stranded DNA for 30 min at 37”. Rat liver DNA had been exhaustively methylated in vitro with unlabelled SAM previously.
The results of our long-term methylation likewise showed that there is no lack of substrate sites in herpesvirus and adenovirus DNA. In contrast to a long-term incubation, in a short-term incubation both viral DNAs accepted only one-half the amount of methyl groups compared with M. luteus DNA using the rat liver DNA methylase and onefifth the amount using the HeLa cell DNA methylase. This could be best explained by a lower binding affinity of eucaryotic methylases for viral DNA. Our competition experiments with viral DNA and cellular DNA in the same assay confirmed a lower affinity of the rat liver DNA methylase for viral DNA compared with cellular DNA and thus offer an explanation of how methylation of viral DNA could be prevented in the cell. ACKNOWLEDGMENTS
We thank M. Klewer and P. Hansen for excellent technical assistence. This work has been supported by the Deutsche Forschungsgemeinschaft. REFERENCES
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58, 55-64.
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157
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