Putative RNA Capping Activities Encoded by Brome Mosaic Virus: Methylation and Covalent Binding of Guanylate by Replicase Protein 1a

The methylated cap structure at the 5′ end of eukaryotic mRNAs is crucial for mRNA stability and efficient initiation of translation. RNA capping in eukaryotic cells proceeds by a series of three reactions, which have been conserved from yeast to mammals (40). mRNA triphosphatase removes the 5′ γ-phosphate of nascent mRNA; then mRNA guanylyltransferase adds a GMP moiety so that a 5′-5′ triphosphate bridge is formed. This reaction proceeds via a covalent enzyme-GMP intermediate. Finally, the capping guanosine moiety is methylated by mRNA guanine-7-methyltransferase to yield the cap structure m7G(5′)ppp(5′)N1pN2p…, which is in some cases further modified by mRNA ribose-O-methyltransferase, acting on the 2′ position of the first two bases. Since capping of cellular mRNAs is a nuclear function closely associated with RNA polymerase II transcription (8, 27), it is not accessible to cytoplasmic viruses. Therefore, many of these encode their own capping systems, some of which conserve the cellular capping reactions. However, various groups of RNA viruses have evolved diverse alternate pathways for cap acquisition (42). The brome mosaic virus (BMV) genome consists of three positive-sense RNA molecules. RNA1 and RNA2 encode proteins 1a and 2a, which are required for RNA replication (25, 45), whereas the 3a protein encoded by the 5′ half of RNA3 is needed for cell-to-cell movement within infected plants (5, 29). A subgenomic RNA4 representing the 3′ end of RNA3 is translated to yield the capsid protein (reviewed in references 1 and 43). All positive-sense BMV RNAs have a 5′ cap structure, m7GpppG (11). BMV RNA replication occurs in 1a- and 2a-containing complexes associated with the endoplasmic reticulum of infected cells (33). The ability of BMV to direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae (22) facilitates studies of multiple aspects of its replication (19, 32, 44). BMV belongs to the large alphavirus-like superfamily of RNA viruses, which in addition to the animal alphaviruses includes the plant bromo-, tobra-, tobamo-, tymo-, carla-, and potexviruses and other groups (2, 15, 23). The hallmark of the alphavirus-like superfamily is conservation of three domains in the RNA replication proteins encoded by these viruses. BMV protein 2a contains one of these domains, which is conserved with RNA-dependent RNA polymerases (17). The other two domains are found in BMV protein 1a (Fig. ​(Fig.1A)1A) (2). These are a DEAD box helicase-related domain comprising the C-terminal half of 1a (16) and a domain implicated in RNA capping at the N terminus of 1a (4, 35). Separating these two domains in 1a is a proline-rich protease-sensitive region (31). In alphaviruses, domains corresponding to the N-terminal and C-terminal halves of BMV 1a are present in nonstructural proteins nsP1 and nsP2, respectively, which are cleaved from a larger precursor by a viral proteinase (Fig. ​(Fig.1A).1A). FIG. 1 Comparison of BMV 1a with alphavirus replicase proteins nsP1 and nsP2. (A) Schematic showing BMV 1a and alphavirus nsP1 and nsP2. The region of most significant similarity between alphavirus nsP1s and the N-terminal half of BMV 1a is shaded (2, 4, 35 ... Two enzymatic activities have been described for alphavirus nsP1. First, guanine-7-methyltransferase, which is able to methylate GTP and dGTP, as well as some 5′-5′ dinucleotides containing guanosine (26, 38). Mutations in Sindbis virus (SIN) nsP1 affecting the methyltransferase activity were found within the nsP1 domain conserved with the N-terminal portion of BMV 1a (28). Based on secondary structure predictions, a large portion of this conserved domain is structurally related to cellular S-adenosylmethionine (AdoMet)-dependent methyltransferases (4). Second, nsP1 forms a covalent complex with m7GMP (3). This complex corresponds to the covalent enzyme-GMP intermediate formed by cellular mRNA guanylyltransferases but is distinct from them in that it contains a methyl group at position 7 of the guanosine ring. Therefore, the covalent m7GMP-nsP1 complex has been proposed to represent an intermediate in a novel pathway for cap formation, where methylation of the capping guanosine precedes its transfer to the 5′ end of mRNA (3). As the domain corresponding to alphavirus nsP1 is only weakly conserved within the superfamily, and in particular between BMV 1a and alphaviruses (Fig. ​(Fig.1B),1B), the degree to which such functions might be conserved in BMV or other alphavirus-like viruses has been uncertain. To explore the possible conservation of these activities within the superfamily and to advance understanding of BMV as a model for positive-strand RNA virus replication, we have tested for similar functions in BMV 1a. Here we show that BMV 1a has guanine-7-methyltransferase and covalent m7GMP binding activities that are similar to those of alphavirus nsP1, though distinct in some particulars.