Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5-triphosphatase activity that specifically removes the ␥ phosphate from the 5 end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m 7 GpppG cap structure was formed at the 5 end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5-triphosphatase activity.Bamboo mosaic virus (BaMV) is a member of the potexvirus group, which belongs to the alphavirus-like superfamily. The ϳ6.4-kb positive-strand RNA genome of BaMV consists of a 94-nucleotide 5Ј-untranslated region, ORF1 (4,098 nucleotides), a triple gene block (ORF2 to ORF4), coat protein-coding region (ORF5), a 142-nucleotide 3Ј-untranslated region, and a poly(A) tail (20). ORF1 of BaMV encodes a 155-kDa polypeptide (replicase) whose amino acid sequence reveals three functional domains: an N-terminal Sindbis virus-like methyltransferase, a central putative RNA helicase, and a C-terminal RNA-dependent RNA polymerase (RdRp) (9,16,24). Recently, the activities of RdRp (18) and RNA capping (guanylyltransferase and methyltransferase) (19) in the C and N termini, respectively, of the BaMV replicase were verified. The central region of the 155-kDa replicase contains several conserved motifs belonging to superfamily 1 (SF1) of RNA helicases (14). This middle region (designated here the helicaselike domain) has thus been hypothesized to be an RNA helicase that assists RdRp in the RNA replication process by unwinding the duplex RNA structure. Besides the central helicase-like domain encoded by ORF1, the 28-kDa movement protein encoded by ORF2 also harbors nucleoside triphosphate (NTP)-binding helicase motifs. Although the overall homology is no more than 20%, the two BaMV proteins have similar sequences in regions containing putative motifs I, II, and VI of SF1 helicases. Since the products of triple gene block are indispensable for the movement of potexviruses through the plasmodesmata between host cells (4, 5), it is believed that the 28-kDa protein helps the viral genome move by its as yet unidentified helicase activity. Recently, the nucleoside triphosphatase (NTPase) and RNA-binding activities on the 28-kDa protein were corrobora...

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.

Bamboo mosaic virus (BaMV) has a 6.4-kb (+) sense RNA genome with a 5′ cap and a 3′ poly(A) tail. ORF1 of this potexvirus encodes a 155-kDa replication protein responsible for the viral RNA replication/transcription and 5′ cap formation. To learn more about the replication complex of BaMV, a protein preparation enriched in the 155-kDa replication protein was obtained from Nicotiana benthamiana by a protocol involving agroinfiltration and immunoprecipitation. Subsequent analysis by SDS-PAGE and mass spectrometry identified a handful of host proteins that may participate in the viral replication. Among them, the cytoplasmic exoribonuclease NbXRN4 particularly caught our attention. NbXRN4 has been shown to have an antiviral activity against Tomato bushy stunt virus and Tomato mosaic virus. In Arabidopsis, the enzyme could reduce RNAi- and miRNA-mediated RNA decay. This study found that downregulation of NbXRN4 greatly decreased BaMV accumulation, while overexpression of NbXRN4 resulted in an opposite effect. Mutations at the catalytically essential residues abolished the function of NbXRN4 in the increase of BaMV accumulation. Nonetheless, NbXRN4 was still able to promote BaMV accumulation in the presence of the RNA silencing suppressor P19. In summary, the replication efficiency of BaMV may be improved by the exoribonuclease activity of NbXRN4.

The S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus belongs to a novel class of mRNA capping enzymes distantly conserved in Alphavirus-like superfamily. The reaction sequence of the viral enzyme has been proposed comprising steps of 1) binding of GTP and S-adenosylmethionine, 2) formation of m 7 GTP and S-adenosylhomocysteine, 3) formation of the covalent (Enzyme-m 7 GMP) intermediate, and 4) transfer of m 7 GMP from the intermediate to the RNA acceptor. In this study the acceptor specificity of the viral enzyme was characterized. The results show that adenylate or guanylate with 5-diphosphate group is an essential feature for acceptors, which can be RNA or mononucleotide, to receive m 7 GMP. The transfer rate of m 7 GMP to guanylate is greater than to adenylate by a factor of ϳ3, and the K m value for mononucleotide acceptor is ϳ10 3 -fold higher than that for RNA. The capping efficiency of the viral genomic RNA transcript depends on the length of the transcript and the formation of a putative stemloop structure, suggesting that mRNA capping process may participate in regulating the viral gene expression.The 5Ј cap structure, m 7 G(5Ј)ppp(5Ј)N, of mRNA serves as a recognition site for ribosome binding in translation and is important for the stability of mRNA against the attack of 5Ј exonuclease in eukaryotic cells. A series of three nuclear enzymatic activities is responsible for the formation of the cap structure (1). First, the ␥-phosphate at the 5Ј end of nascent mRNA is removed by RNA 5Ј-triphosphatase; second, the GMP moiety of GTP molecule is transferred to the 5Ј-diphosphate end of the RNA via a 5Ј-5Ј linkage by guanylyltransferase; and finally, a methyl group is added to N7 of the transferred guanylate from S-adenosylmethionine (AdoMet) 1 by methyltransferase. The cap formation pathway for viruses within the Alphavirus-like superfamily differs from the nuclear mechanism in that methylation of GTP occurs before transguanylation; in other words, m 7 GMP, rather than GMP, is transferred to the 5Ј end of the 5Ј-diphosphate RNA during the cap formation process. This distinctive mRNA capping reaction is catalyzed by a group of distantly conserved AdoMet-dependent guanylyltransferases that were identified in Semliki Forest virus (2), hepatitis E virus (3), tobacco mosaic virus (4), brome mosaic virus (5, 6), and bamboo mosaic virus (BaMV) (7). Together with other characteristics such as their membrane-association nature and distinctive protein primary structures, this class of enzyme represents a novel mRNA capping enzyme different from those evolutionally conserved guanylyltransferases and methyltransferases in DNA viruses, metazoans, and fungi. Although the critical roles of the conserved amino acids in the enzymatic activities of the enzyme of Semliki Forest virus (8) and BaMV (9) have been addressed, the molecular mechanism of this class of enzyme is far from being understood.BaMV, a member of Alphavirus-like superfamily, has a ϳ6.4-kilobase positive-strand RNA genome with a cap s...

The helicase-like domain of BaMV replicase possesses NTPase and RNA 5'-triphosphatase activities. In this study, mutational effects of the helicase signature motifs and residue L543 on the two activities were investigated. Either activity was inactivated by K643A-S644A, D702A, D730A, R855A, or L543P mutations. On the other hand, Q826A, D858A and L543A had activities, in terms of k(cat)/K(m), reduced by 5- to 15-fold. AMPPNP, a nonhydrolyzable ATP analogue, competitively inhibited RNA 5'-triphosphatase activity. Analogies of mutational effects on the two activities and approximation of K(i(AMPPNP)) and K(m(ATP)) suggest that the catalytic sites of the activities are overlapped. Mutational effects on the viral accumulation in Chenopodium quinoa indicated that the activities manifested by the domain are required for BaMV survival. Results also suggest that Q826 in motif V plays an additional role in preventing tight binding to ATP, which would otherwise decrease further RNA 5'-triphosphatase, leading to demise of the virus in plant.