Vol. 23, No. 1 Printed in U.S.A.

JOURNAL OF VIROLOGY, July 1977, p. 110-116 Copyright 0 1977 American Society for Microbiology

Methyl Group Analysis of Virion-Associated High-MolecularWeight RNA Synthesized In Vitro by Purified Vaccinia Virus DONALD L. NUSS* AND ENZO PAOLETTI Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201 Received for publication 24 January 1977

The methylation pattern of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus has been determined. Analysis of purified high-molecular-weight RNA synthesized with S-[methyl-3H]adenosylmethionine and a-[32P]UTP as precursors gave the following results. (i) Essentially all molecules contained blocked and methylated structures of the type m7G(5')ppp(5')G"n and m7G(5')ppp(5')Am. (ii) There was no detectable methylation at internal sites. (iii) Under several different conditions of synthesis, the ratio of molecules containing m7G(5')ppp(5')Gm to those containing m7G(5')ppp(5')Am was similar for both the virion-associated high-molecularweight RNA and the virion-released 8-12S mRNA.

The in vitro synthesis and partial characteri- min at 39,000 x g. The virus pellet containing the zation of high-molecular-weight (24-26S) vir- virion-associated RNA was resuspended by sonic ion-associated RNA of vaccinia virus has re- treatment in 50 mM Tris-hydrochloride (pH 8.4), 20 EDTA, and 10 mM dithiothreitol. Both this cently been reported (17). In a previous paper, mM fraction and the virus-free supernatant containing data compatible with a precursor-product relavirion-released RNA were made 0.5% with retionship between this RNA and virion-released the spect to sodium dodecyl sulfate (SDS) and were then 8-12S vaccinia mRNA were presented (18). extracted, using the hot phenol-SDS method deThe 5'-terminal end of vaccinia virus mRNA scribed by Girard (8). RNA was precipitated from synthesized and released from the virion in the final aqueous phase with ethanol, followed by vitro is known to have the structures m7G- three cycles of precipitation with 2 M LiCl (2). The virion-associated and virion-released RNA (5')ppp(5')Gm and m7G(5')ppp(5')Am (24). Considerable progress in elucidating the mecha- fractions were layered on 5 to 20% sucrose gradients mM sodium acetate (pH 5.1), 100 mM NaCl, nism of formation of these 5'-terminal caps has in 100.1% SDS and sedimented as previously debeen forthcoming (6, 13, 14). Although the vir- and scribed (17). Preheating of the RNA samples (1000C ion-associated high-molecular-weight RNA is for 20 s) followed by quenching in ice prior to sedialso methylated (18), the nature and extent of mentation greatly reduced aggregation of RNA on methylation has not previously been reported. the gradient. Pooled fractions from the SDS-sucrose This information is necessary for a full under- gradients were precipitated with ethanol, and the standing of the origin, function, and fate of the resulting pellets were dissolved in a minimal volume of 10 mM Tris (pH 7.6) and 0.1 M NaCl, folhigh-molecular-weight RNA species.

lowed by a stepwise addition of 9 volumes of dimethyl sulfoxide (Me2SO). They were then heated for 2 min at 60°C and sedimented through a 0 to 15% sucrose gradient in 99% Me2SO, 10 mM LiCl, and 0.5 mM sodium EDTA (pH 7.1), using an SW41 rotor at 39,000 rpm for 26 h. Selected fractions were pooled, and the RNA was precipitated with ethanol and resedimented on a second 5 to 20% SDS-sucrose gradient. Enzyme digestions. Purified RNA fractions were precipitated by ethanol, and the resulting pellet was dissolved in 100 ,ul of 5 mM sodium acetate, pH 5.8. They were digested twice, first for 2 h at 37°C with 500 ,ug of Penicillium nuclease per ml and then for an additional hour with 20 U of bacterial alkaline phosphatase per ml at pH 8. The mixture of m7G(5')ppp(5')Gmn and m7G(5')ppp(5')Am recovered after paper electrophoresis

MATERIALS AND METHODS Synthesis of vaccinia virus RNA. Purified vaccinia virus particles (strain WR) were prepared from infected HeLa cells as described by Joklik (10). A typical 20-ml reaction mixture contained 110 optical density (at 260 nm) units of purified virus; 50 mM Tris-hydrochloride (pH 8.4); 10 mM MgCl2; 10 mM dithiothreitol; 0.05% Nonidet P-40; 2 mM each ATP, GTP, and CTP; 0.75 mCi of a-[32P]UTP (final specific activity, 37.5 mCi/mmol); and 0.4 mCi of neutralized S-[methyl-3Hladenosylmethionine, 8.9 Ci/mmol. Incubation was for 40 min at 37°C. Purification of virion-associated high-molecularweight and virion-released 8-12S RNA. The transcription reaction was terminated by adding Na3EDTA to a final concentration of 20 mM, and virus particles were pelleted by centrifugation for 45 110

VOL. 23, 1977

HIGH-MOLECULAR-WEIGHT RNA METHYLATION PATTERN

was digested with 0.3 U of nucleotidyl pyrophosphatase for 1 h at 370C in 20 mM Tris-hydrochloride (pH 7.5) and 1.5 mM magnesium acetate. This material was further digested by bacterial alkaline phosphatase for an additional 30 min in the same buffer. Paper electrophoresis and chromatography. Samples to be analyzed were applied in 3-cm strips onto Whatman 3MM paper and subjected to electrophoresis at 2,600 V for 60 min in pyridine acetate buffer, pH 3.5 (15). The positions of authentic compounds were located under UV light, and the paper strips were cut into 1-cm fractions for counting in scintillation fluid. Paper chromatography was performed with isopropanol, water, and concentrated ammonium hydroxide (7:2:1) (14). Fractions (1 cm) were soaked in 1 ml of 1 M LiCl and counted in 10 ml of Aquasol scintillant. of Sources materials. S - [methyl -3HIadenosylmethionine was purchased from Amersham/Searle and a-[32P]UTP was purchased from New England Nuclear Corp. Ribonucleoside triphosphates, 2'-O-methylguanosine, 2'-O-methyladenosine, and 7-methylguanosine, were purchased from P-L Biochemicals. Bacterial alkaline phosphatase was purchased from Worthington Biochemicals Corp., and nucleotide pyrophosphatase and Penicillium nuclease were purchased from Sigma Chemical Co. and Yamasa Shoyu Co., respectively.

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RESULTS Purification of the virion-associated highmolecular-weight and virion-released 8-12S RNA. A different sedimentation profile was observed for a- [32P]UMP- and [3Hlmethyl-laFion-reasediRNtAtiondcthrug th 2.5 methyl beled accinia virus RNA extracted from the of [3H]methyl- and a-[32P]UMP-labeled RNA. resuspended virus pellet than was observed for dients (A) Virion-released RNA. (B) Virion-associated RNA extracted from the virus-free reaction RNA. (0) ratio of 3H to 32)P incorporation mixture supernatant (Fig. 1). The virion-re- across Symbols: the gradient; (@) [PH~methyl cpm; (Q) 32P~ leased RNA sedimented as a narrow band be- cpm. tween the 4S and 18S rRNA markers, with a radioactivity peak between 8S and 12S. The the high-molecular-weight RNA is always assovirion-associated RNA fraction contained not ciated with the virus pellet; i.e., the virus-free only RNA sedimenting at 8-12S, but also faster- supernatant contains 8-12S RNA, whereas the sedimenting RNA with sedimentation values virus pellet contains both 8-12S and the fasteras high as 28-30S, as previously reported sedimenting RNA species. The[3t imethylo to a- inP]UMPratio for vir(17, 18). The proportion of labeled RNA that was ex- ion-released RNA indicated that 2.2 methyl tractable from the virus pellet, as opposed to groups were incorporated per 1,000 nucleotides, the virus-free reaction mixture supernatant, consistent with the data of Wei and Moss (24). varied from experiment to experiment. When These workers reported that the methyl groups virus was pelleted for 30 min at 25,000 x g, the are incorporated exclusively into the 5'-termipercentage of radioactive RNA extracted from nal ends of the virion-released 8-12S mRNA in the supernatant varied from as much as 80% to the form Of m7G(5')ppp(5')Glh and m7Gas low as 20% of the total extractable RNA. (5')ppp(5')Am. The nearly linear decrease in the Under the conditions used in Fig. 1 (39,000 x g ratio of 3H to 32P with the increasing sedimentafor 45 min), the amount of extractable RNA in tion values (Fig. 1A) is consistent with all molthe supernatant was usually on the order of 10 ecules being capped at the 5'-terminal end. The to 30% of the total. The precise nature of this deviation in slope for this ratio in fractions 12 variation is not well understood and requires through 16 (Fig. iB) suggested the possibility of further investigation. It is clear, however, that a small degree of RNA-RNA aggregation or

112

NUSS AND PAOLElIJJ. VIROL.

methylation of the large RNA at sites in addition to the cap. In this regard, before the methyl constituents were analyzed, the possibility of RNA-RNA aggregation was eliminated by denaturing the RNA fractions recovered from the SDS-sucrose gradients in 90% Me2SO and subsequent sedimentation through a sucrose gradient in 99% Me2SO. Fractions I', I, and II (in Fig. 1) sedimented in a similar fashion with respect to the marker rRNA in both the SDS-sucrose and the sucrose-99% Me2SO gradients. For fraction III, however, 85% of the radioactivity sedimented with a mean value of 248, whereas 15% sedimented as 8-12S RNA after denaturation (not shown). Portions of the fractions recovered from the sucrose-Me2SO gradients were resedimented on a second SDS-sucrose gradient (Fig. 2). The profiles show that a clear separation of 8-12S RNA from the high-molecular-weight RNA was achieved. Although the 8-12S profile shown is for the fraction purified from the virion-associated RNA, virion-released 8-12S RNA gave an identical sedimentation profile with a slightly higher 3H/32P ratio (Table 1). Analysis of methyl constituents. The purified RNA fractions were subjected to Penicillium nuclease and bacterial alkaline phosphatase digestion and analyzed by paper electrophoresis in pyridine acetate buffer (pH 3.5). The results (Fig. 3) show that 99% of the

[3Hlmethyl radioactivity migrated between the pA and pG markers in the position expected for m7G(5')ppp(5')G" and m7G(5')ppp(5')Am (24). In each preparation a small amount (