Purification of mRNA Guanylyltransferase and mRNA(guanine-7-)methyltransferase from Vaccinia Virions

The sequences m7G(5’)pppGm-and m’G(5’)pppA”-are located at the 5’ termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5’ terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA(guanine-7-)methyl-transferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl-and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an s,,,,~ o f 6.0, and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl

The sequences m7G(5')pppGm-and m'G(5')pppA"-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase.
The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA(guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl-and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an s,,,,~ o f 6.0, and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations.
Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system.
Unique methylated structures have been identified at the 5' terminus of viral mRNAs (l-6), viral genome RNA (7), and mammalian cellular mRNAs (8)(9)(10)(11). These structures consist of a terminal 'I-methylguanosine that is attached to a penultimate ribosemethylated nucleoside via a triphosphate bond and may be represented as the sequence m7G(5')pppNm-.I It is evident that such structures cannot be formed by the usual transcriptive mechanism. Vaccinia, a DNA virus that replicates within the cytoplasm of infected cells, is an ideal system for determining the enzymatic formation of these structures. Messenger RNA synthesized in vitro in the presence of S-adenosylmethionine by the vaccinia core-associated RNA nucleotidyltransferase has the 5'-terminal sequences m'G(5')pppGm-and m'G(5')pppAm- (1). Furthermore, disruption of the vaccinia core releases the activities which are capable of completely modifying the 5' termini of unmethylated vaccinia mRNA (12). Three activities have been identified: mRNA guanylyltransferase, mRNA(guanine-'I-)methyltransferase, and mRNA(nucleoside-2'-)methyltransferase. In this paper we present the purification and physical characterization of the activities which catalyze the transfer of GMP to RNA and the methylation of the added terminal * Present address, New York State Department of Health, Division of Laboratories and Research, Albany, N.Y. 12201.

EXPERIMENTAL PROCEDURES
Virus-Vaccinia virus (strain WR) was purified from infected HeLa cells by sedimentation through a sucrose cushion and two subsequent sucrose gradient sedimentations as previously described (14,15). Radioactive virus was grown in the presence of ['Hlleucine and was purified in a similar manner. Preparation of mRNA Substrate-Vaccinia virus in vitro mRNA was prepared from 500-ml reaction mixtures which contained 500 A,,, units of vaccinia virus, 50 rnM Tris-HCl (pH 8.4), 10 rn~ dithiothreitol, 0.05% Nonidet P-40, 7.5 rn~ MgCl*, 3.75 mM ATP, and 1.25 mM each GTP, CTP, and UTP. Following incubation for 2 hours at 37", viral cores were removed by centrifugation at 25,000 x g in a Sorvall SS-34 rotor for 30 min at 4". From the pooled supernatants the RNA was precipitated with cetyltrimethylammonium bromide according to the procedure of Sibatani (16), and was collected by centrifugation at 12,000 x g in a Sorvall HB-4 rotor for 20 min at 4". The pooled RNA pellets were dried under an air stream, dissolved in absolute ethanol, and precipitated as the sodium salt from 0.1 M sodium acetate/70% ethanol overnight at -20°. Following ethanol precipitation the RNA was purified further by passage through a column (55 x 0.9 cm) of Sephadex G-50. The yield of RNA was 5 mg.
A procedure for the preparation of synthetic poly(A) that is a suitable acceptor for the guanylyltransferase is described in the accompanying paper (13 DNA-cellulose was less satisfactory because the activities apparently adsorbed to the cellulose matrix.
After determining that the enzymes did not bind to DNAagarose we decided to use RNA-affinity columns as selective steps for further purification.
The flow-through of the DNAagarose column was applied directly to a column (10 x 0.6 cm) of poly(U)-Sepharose equilibrated with Buffer A containing 0.05 M NaCl. Following a wash with this same buffer, a linear gradient from 0.05 to 0.5 M NaCl in Buffer A was applied to the column. Fractions were collected and assayed for guanylyland methyltransferase activities, and in the experiments in which [BH]leucine-labeled virus was used, portions were counted for total protein. Fig. 1 illustrates a typical profile of the elution of total protein and guanylyl-and methyltransferase activities from poly(U)-Sepharose.
Approximately 80% of the applied protein eluted in the flow-through.
Ten per cent of the applied protein eluted at 0.36 M NaCl and was coincident with the major peak of both guanylyl-and methyltransferase activities.
No further elution of either protein or enzyme activities was detected at salt concentrations greater than 0.5 M. Variable but always minor amounts of both guanylyl-and methyltransferase activities eluted at 0.25 M NaCl. The properties of the activities eluting at this ionic strength do not differ from those in the major peak of activity, except for their lower specific activities. This is due to the presence of other proteins eluting in this region as demonstrated by gel electrophoretic analysis of the fractions (data not shown).
The entire major peak of activities from the poly(U)-Sepharose column was pooled, dialyzed against Buffer A containing 0.05 M NaCl, and applied to a column (5 x 0.6 cm) of poly(A)-Sepharose equilibrated with Buffer A containing 0.05 M NaCl. The column was washed with the same buffer, and a linear gradient from 0.05 to 0.4 M NaCl in Buffer A was applied to the column. Fractions were collected and assayed as [SH]Leucine-labeled protein (---) was 33% Triton X-100. determined by counting 0.1 ml of each fraction.
described for the poly(U)-Sepharose column. The elution of total protein and the enzyme activities from poly(A)-Sepharose is shown Fig. 2. Only two major peaks of protein are present. The peak eluting at 0.17 M NaCl is coincident with both guanylyl-and methyltransferase activities, whereas the second protein peak elutes in a region of diminishing enzyme activities. A 2-fold increase in specific activity is achieved in this step. Chromatography on either phosphocellulose, CM-Sephadex, or DEAE-cellulose resulted in no additional purification of the activities.
For experiments with purified enzyme, fractions around the peak of activity on poly(A)-Sepharose comprising approximately two-thirds of the total activity were pooled (e.g. Wash Gradient I 1  Fig. 2). The enzyme has been stored for several months at 4' or -20" without loss of activity. A summary of the purification of the guanylyl-and methyltransferase activities from vaccinia virus is presented in Table  I. Following high speed centrifugation of the disrupted viral cores 17% of the initial viral proteins are recovered. In this step there is a g-fold increase in specific activity and a 150% recovery of the methyltransferase which may result from an increased accessibility of the enzyme to the substrates as it is solubilized from the core particle. By contrast, a 2.4-fold increase in specific activity and an apparent 40% recovery of the guanylyltransferase is observed in this step. One explanation for this is that the vaccinia core-associated nucleoside triphosphatase, capable of hydrolyzing GTP to GDP in the presence of nucleic acid (28), is also released in this step. The hydrolysis of GTP would produce artificially low values for the recovery of the guanylyltransferase.

Fractions 35 to 41 in
Passage of the material through DNA-agarose, onto which the nucleoside triphosphatase adsorbs, results in full recovery of the guanylyltransferase activity and a 17-fold increase in its specific activity. At this stage only 70% of the methyltransferase is recovered; however, as is discussed in the following section this loss of total methyltransferase activity is due to removal of the mRNA(nucleoside-2'-)methyltransferase.
Indeed the separation of the methyltransferases is accomplished most effectively in this step. Further increases in specific activities of both guanylyland methyltransferases are achieved by chromatography on poly(U)-Sepharose and poly(A)-Sepharose. The recovery of the guanylyl-and methyltransferase activities after these steps is 54 and 17%, respectively, of that observed in the detergenttreated virus. Following poly(A)-Sepharose chromatography, 0.4% of the initial viral protein remains, and there is an ap-9325 parent 150-fold purification of the guanylyltransferase and 50fold purification of the methyltransferase. An additional increase in the specific activities of the guanylyl-and methyltransferase activities is observed in the peak fraction from poly(A)-Sepharose chromatography corresponding to an ultimate 235-fold and 106-fold purification of these activities, respectively.
Since the virus contains two separate methyltransferase activities is observed in the peak fraction fro& mRNA(guanine-7-)methyltransferase, as shown below, the true purification achieved is nearly twice the indicated value. Identification of Purified Methyltransfemse-The nucleoside products formed by the methyltransferase at various stages of purification were analyzed by thin layer chromatography after the RNA was digested completely with ribonucleases A and T1, followed by snake venom phosphodiesterase and alkaline phosphatase.
Viral cores exhibit mRNA(guanine-7-)methyltransferase, mRNA(adenosine-2'-)methyltransferase, and mRNA(guanosine-2'-)methyltransferase activities as indicated in Fig. 3A. Sixty-six per cent of the methyl groups are incorporated into 7-methylguanosine by the methyltransferases associated with the viral cores, and the remainder of the methyl groups are incorporated into the 2'-O-methylribonucleosides. The ratio of the mRNA(guanine-7-)methyltransferase activity to the mRNA(nucleoside-2'-)methyltransferase activity increases during the subsequent steps of purification as shown in Panels 23 through D in Fig. 3. After poly(U)-Sepharose chromatography 99% of the methyl groups are incorporated by the enzyme into 7-methylguanosine. Thus the purified enzyme exhibits, in addition to guanylyltransferase activity, mRNA(guanine-7-)methyltransferase activity but no significant mRNA(nucleoside-2'-)methyltransferase activity. phoresis-Samples taken at various stages of the purification procedure were analyzed for their polypeptide composition on 7.5% polyacrylamide gels containing 5 M urea and 0.1% sodium dodecyl sulfate. Fig. 4 shows the polypeptide composition of whole virions, viral cores, flow-through from the second DEAEcellulose column, flow-through from the DNA-agarose column, and the pooled enzyme fractions from the poly(U)-Sepharose column in Gels A through E, respectively. In Gel E only one major polypeptide is clearly seen in the photograph although additional minor bands were evident on visual inspection. Gel electrophoretic analysis of individual fractions from the poly(A)-Sepharose column suggested that the major band and one minor band were associated with enzyme activity. The stained gels in Fig. 5  in Gel E which cannot be observed in this photograph. Electrophoresis was from top to bottom. did not. This was confirmed by gel electrophoretic analysis of the fractions from the poly(A)-Sepharose column during purification of the enzyme from [SH]leucine-labeled virus. The distribution of radioactivity in one sliced gel is shown in Fig. 6. The ratio of the amounts of the radioactivity in the two more abundant polypeptides was constant across the peak of enzyme activity. By contrast, the ratio of the radioactivity in the very minor polypeptide with respect to either of the other two polypeptides decreased across the peak of enzyme activity suggesting it was a contaminant.
The molecular weights of the two enzyme-associated polypeptides were determined on 7.5% polyacrylamide gels containing 5 M urea and 0.1% sodium dodecyl sulfate. By comparison with the mobilities of polypeptides of known molecular weights (29,30) values of 35,000 and 31,400 were derived for the two polypeptides. Using these molecular weights and assuming similar leucine compositions of both polypeptides, the molar ratio of the larger to the smaller polypeptide is 0.7. Alternately, when the molar ratio for these same polypeptides was determined from the area under curves of densitometric tracings of stained gels, the ratio 1. their s~,,~ was determined to be 6.0. Determination of Molecular Weights-Siegel and Monty have described the applicability of determining the molecular weights of proteins from their sedimentation coefficients and Stokes radii (32). From these parameters determined by sucrose gradient sedimentation and gel filtration, and assuming a partial specific volume of 0.73 cmS/g, a value of 127,000 was obtained for the molecular weight of the guanylyl-and methyltransferase.
Additionally These activities are inseparable by affinity or ion exchange chromatography, gel filtration, and sucrose density gradient sedimentation.
The purified enzyme has a molecular weight of 127,000 and contains two polypeptides whose molecular weights are 95,000 and 31,400 and whose molar ratios are close to unity suggesting the mRNA guanylyltransferase and mRNA(guanine-7-)methyltransferase are components of an enzyme system. 44  Although the mRNA guanylyltransferase and mRNA(guanine-7-)methyltransferase isolated from vaccinia virus cores appear to be components of a single enzyme system, the mRNA(nucleoside-2'-)methyltransferase is a different enzyme. It is separated from the mRNA guanylyl-and (guanine-7-)methyltransferase during the purification by its affinity for DNA-agarose.
The modified Y-terminal sequences of mRNAs from a variety of viruses and mammalian cells are similar to those found in vaccinia mRNA. Therefore, analogous enzymes must be present in cells and other viruses which perform this modification.
Thus far, only the vaccinia enzyme has been purified, enabling us to study the process of modification of the 5' terminus of mRNA in vitro. The characterization of the reactions catalyzed by the mRNA guanylyltransferase and the mRNA(guanine-7-)methyltransferase is presented in the following paper (13).
Upon entry of vaccinia virus into the cytoplasm of the host cell the RNA nucleotidyltransferase (RNA polymerase) associated with the viral core (38, 39) is responsible for viral mRNA synthesis. Of the enzymes successfully isolated from vaccinia virus cores, the poly(A) polymerase (27) and the mRNA guanylyltransferase and mRNA(guanine-7-)methyltransferase are involved clearly in the modification of RNA. The biological roles of two nucleic acid-dependent nucleoside triphosphate phosphohydrolases (24,28), a single strand-specific deoxyribonuclease (25,40), and a protein kinase (26,41) are obscure. It seems reasonable, nevertheless, to consider that some of these enzymes may be involved in transcription.
For example, Kates and Beeson have proposed that in vaccinia virus there may be an association between the adenosine triphosphatase and the ATP-dependent extrusion of mRNA from viral cores (42). In Escherichiu coli p factor, which is responsible for termination of RNA synthesis, has been shown to possess RNA-dependent nucleoside triphosphate phosphohydrolase activity (43). Additionally, a bacteriophage T7-induced protein kinase which can phosphorylate the B' subunit of the host cell RNA nucleotidyltransferase appears to be important in regulation of transcription (44,45).
Although proof that the enzymes within the vaccinia core are encoded by the viral genome is lacking, the RNA nucleotidyltransferase (46,47), deoxyribonuclease (48), nucleoside triphosphate phosphohydrolases (49,50), and poly(A) polymerase (51) are all induced after viral infection. Preliminary experiments% suggest that HeLa cell mRNA(guanine-'l-)methyltransferase exhibits different chromatographic properties from the vaccinia virus core-associated enzyme indicating that the latter is also viral-induced.