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Virology 259, 200–210 (1999) Article ID viro.1999.9763, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector The N-Terminal Half of the Brome Mosaic Virus 1a Protein Has RNA Capping-Associated Activities: Specificity for GTP and S-Adenosylmethionine F. Kong, K. Sivakumaran, and C. Kao1 Department of Biology, Indiana University, Bloomington, Indiana 47405 Received February 8, 1999; returned to author for revision March 29, 1999; accepted April 14, 1999 The N-terminal half of the brome mosaic virus (BMV) 1a replication-associated protein contains sequence motifs found in RNA methyltransferases. We demonstrate that recombinant BMV methyltransferase-like (MT) domain expressed in Esche- richia coli forms an adduct with a guanine nucleotide in a reaction that requires S-adenosylmethionine (AdoMet) and divalent cations. Moieties in GTP and AdoMet required for adduct formation were determined using a competition assay and chemical analogues. In the guanine nucleotide the ribose 29 hydroxyl, the triphosphates, the base C6 keto group, and possibly the N1 imine are required. In AdoMet, the methyl group and the ability to transfer a methyl group to guanine nucleotide were demonstrated to be required for adduct formation. The effects of methyltransferase inhibitors on viral RNA synthesis was determined using an in vitro RNA synthesis assay. These results are consistent with the previously reported activities of alphaviral nsP1 methyltransferase protein and identify the chemical moieties required for the BMV methyltransferase activity. © 1999 Academic Press INTRODUCTION Capping-associated guanylyltransferase and methyl- transferase activities have not yet been reported for BMV. The translation and stability of mRNA are enhanced by We have been characterizing the activities of the BMV 9 9 the addition of a 7-methyl-GMP cap through a 5 -5 replicase for RNA synthesis and viral protein–protein linkage to a transcript (for review, see Shuman and interactions. Using the yeast two-hybrid system, O’Reilly Schwer 1995). Capping of cellular mRNAs occurs in the et al. (1997) reported that 1a residues 1–516 contain a nucleus. However, many viruses replicate in the cyto- domain sufficient for interaction with the corresponding plasm and hence may code for their own capping activ- N terminus of another 1a subunit. Computer-predicted ities. Several viral guanylyltransferase and methyltrans- secondary structures within these 516 residues revealed ferases have been identified and characterized, includ- a high degree of similarity in several alphaviral species ing those from Sindbis virus (Mi and Stollar, 1991), (O’Reilly et al., 1998). Furthermore these secondary struc- Semliki Forest virus (Laakkonen et al., 1994); reovirus tures are similar to the known structures of the DNA (Mao and Joklik, 1991), rotavirus (Pizarro et al., 1991), methyltransferase, HhaI (O’Reilly et al., 1998). In this Bluetongue virus (Le Blois et al., 1992), vaccinia virus communication, we expressed the N-terminal 516 amino (Martin and Moss, 1975, 1976), and others. Viral and acids of the BMV 1a protein in E. coli and assay for a cellular capping enzymes have mechanistically distinct classic guanylyltranserase activity, the formation of a activities. For example, the alphaviral nsP1 capping pro- covalent intermediate with the guanine nucleotide. After tein methylates GTP to form 7-methyl-GTP (m7GTP) prior establishing this activity, we analyzed the chemical moi- to its transfer to viral RNA, whereas the cellular capping eties in GTP and the methyl donor, S-adenosylmethi- activity transfers the guanine nucleotide to the RNA prior onine (AdoMet) that are required for the formation of a to the methyltransfer reaction. protein-guanine nucleotide intermediate. Brome mosaic virus (BMV) is a plant-infecting member of the alphavirus-like superfamily (Koonin, 1993). The RESULTS BMV RNA genome consists of three capped RNAs. The longest RNA, RNA1 codes for the 1a protein, which has G-1a and G-MT expression sequence homologies to RNA capping and helicase ac- To facilitate the purification of the 1a MT-like domain, we tivities in its N- and C-terminal halves, respectively (Ha- fused the cDNA coding for the full-length or the N-terminal seloff et al., 1984; Ahlquist, 1992; Rozanov et al., 1992). 516 residues of 1a to the GST-coding sequence. The ex- pected fusion protein should be of 136 kDa for full-length 1a (G-1a), and 75 kDa for the N-terminal MT domain (G-MT). 1 To whom reprint requests should be addressed. Fax: (812) 855- After enrichment in batch through glutathione beads, bands 6705. E-mail: [email protected]. of the expected mass were the predominant products in 0042-6822/99 $30.00 Copyright © 1999 by Academic Press 200 All rights of reproduction in any form reserved. RNA CAPPING-ASSOCIATED ACTIVITIES 201 FIG. 1. Expression and activity of the BMV capping domain. (A) Coomassie Blue-stained 10% PAGE–SDS containing the supernatant of a 15,000 g (S15)-clarified bacterial lysate and the eluant from glutathione-agarose column. Sizes of the molecular mass markers in kilodaltons (kDa) are indicated on the left of the gel. The identities of the enriched proteins, glutathione S-transferase (GST), and GST fused to the full-length 1a (G-1a) and the N-terminal 516 residues of the 1a protein (G-MT) are indicated on the right. Lane numbers are on the bottom of the autoradiograph. (B) Ability of the BMV capping domain to form a covalent adduct with guanine nucleotide. The S15 and eluant fractions in (A) were incubated with buffer B, whose 32 components include 4 mCi [a- P]GTP, 100 mM AdoMet, and 2 mM MgCl2. The samples were analyzed by SDS–PAGE and autoradiography. The expected positions of the three recombinant proteins are indicated on the left. gels stained with Coomassie Blue (Fig. 1A). In Western protein are essential for binding guanine nucleotide. blots, these products reacted against serum specific for Furthermore the BMV methyltransferase-like domain is GST (F. Kong, data not shown). In the preparations contain- required for guanine nucleotide binding because GST ing G-MT, some bands lower in molecular weight than 75 alone was unable to form an adduct with radiolabeled kDa were also recognized by anti-GST, indicating that they guanine nucleotide (Fig. 1B, lane 4). G-MT that had been contain truncations of the C-terminal BMV MT domain (data treated with thrombin to separate the BMV MT from the not shown). GST also bound GTP (F. Kong, data not shown). However, The nsP1 proteins of Sindbis (SIN) and Semliki Forest the cleavage reaction did not work efficiently, and we virus (SFV) can form a covalent intermediate with a meth- routinely assayed for guanine nucleotide binding with ylated GMP that will survive denaturing protein gel elec- the fusion protein. trophoresis (Laakkonen et al., 1994; Ahola and Ka¨a¨ri- G-1a did not form adduct with radioactive guanine a¨inen, 1995). To determine whether G-1a and G-MT can nucleotide significantly above background levels despite form a adduct with a guanine nucleotide, we incubated the fact that it was present in higher abundance than clarified lysate S15 and the glutathione-enriched fractions G-MT in the S15 fractions (Fig. 1B, lanes 2 and 5). Low- for 30 min with [a-32P]GTP, AdoMet, and magnesium. level binding to radiolabeled guanine nucleotide was After stopping the reaction by the addition of Laemmli observed in some experiments, but all at much reduced sample buffer containing 1% SDS, the sample was level on a molar basis in comparison with G-MT. Several heated to 90°C and then electrophoresed in a denaturing independently generated G-1a preparations behaved in polyacrylamide gel. A parallel gel containing the same a similar manner, suggesting that the lack of guanine protein samples was stained with Coomassie Blue to nucleotide binding is not due to an unexpected mutation visualize the proteins. Autoradiography revealed a band in 1a. As shall be presented later, 1a present in the BMV identical in position to G-MT (Fig. 1B, lanes 3 and 6). replicase extracted from infected plants does have gua- Faintly labeled products seen in the S15 fractions were nine nucleotide-binding activity. absent in the preparations containing the more pure GST-enriched fractions containing G-MT. In these reac- Requirements for the formation of the G-MT-guanine tions (Fig. 1B, lane 6), two bands of ,75 kDa also formed nucleotide covalent adduct an adduct with guanine nucleotide (Fig. 1B, lane 6). Because this truncated protein contains an N-terminal To define the requirements for the G-MT-guanine nu- GST as determined by Western blot analysis (F. Kong, cleotide adduct, we examined the effects of different data not shown), not all of the first 516 residues of the 1a components in the binding reaction (Fig. 2). In the pres- 202 KONG, SIVAKUMARAN, AND KAO are similar to those seen with the alphaviral nsP1 protein (Ahola and Ka¨a¨ria¨inen, 1995). Guanine nucleotide moieties required to interact with G-MT G-MT could bind [a-32P]-GTP but not [a-[32P]ATP] (Fig. 3A, lanes 2 and 5), indicating that it has specificity for guanine nucleotide. To identify the chemical moieties in GTP required for specific recognition, we added an in- creasing amount of different nucleotides competitors to a reaction containing constant amounts of M-GT and [a-32P]GTP. The effect on adduct formation was then quantified after gel electrophoresis. With unlabeled GTP as the competitor, the amount of radiolabeled adduct was reduced in a concentration-dependent manner (Fig. 3B). In contrast, ATP, CTP, UTP, dATP, dCTP, and dTTP did not significantly reduce the amount of the G-MT-guanine nucleotide adduct at even a 500-fold molar excess of the FIG.

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