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 γ 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), a member of the potexvirus group, infects primarily members of the Bambusoideae . Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5′ end of the viral genome. To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in Saccharomyces cerevisiae . All three versions of BaMV ORF1 proteins could be radiolabeled by [α- ³² P]GTP, which is a characteristic of guanylyltransferase activity. The presence of S -adenosylmethionine (AdoMet) was essential for this enzymatic activity. Thin-layer chromatography analysis suggests that the radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 protein is m ⁷ GMP. The N-terminal domain also exhibited methyltransferase activity that catalyzes the transfer of the [ ³ H]methyl group from AdoMet to GTP or guanylylimidodiphosphate. Therefore, during cap structure formation in BaMV, methylation of GTP may occur prior to transguanylation as for alphaviruses and brome mosaic virus. This study establishes the association of RNA capping activity with the N-terminal domain of the replicase of potexviruses and further supports the idea that the reaction sequence of RNA capping is conserved throughout the alphavirus-like superfamily of RNA viruses.

A gene encoding 1,3-β-glucanase was isolated from Streptomyces sioyaensis based on an activity plate assay. Analysis of the deduced amino acid sequence of the gene revealed that the matured 1,3-β-glucanase has two functional domains separated by a stretch of nine glycine residues. The N-terminal domain shares sequence similarity with bacterial endo-1,3-β-glucanases classified in glycosyl hydrolase family 16 (GHF 16), while the C-terminal domain is a putative carbohydrate-binding module (CBM) grouped into CBM family 6. To characterize the function of each domain, both the full-length and the CBM-truncated versions of the protein were expressed in Escherichia coli and purified to homogeneity. Biochemical data suggest that the glycosyl hydrolase domain preferentially catalyses the hydrolysis of glucans with 1,3-β linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble β-glucans (1,3-, 1,3–1,4- and 1,4- linkages) but not to xylan, a primary binding target for most members of CBM family 6. The full-length and the CBM-truncated proteins had similar specific activity (units per mol of hydrolase domain) on soluble 1,3-β-glucan, whereas the former had much stronger specific activity on insoluble 1,3-β-glucans, suggesting that the C-terminal CBM enhances the activity of the S. sioyaensis 1,3-β-glucanase against insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrate.

Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae . The open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that was postulated to be involved in the replication and the formation of cap structure at the 5′ end of the viral genome. To characterize the activities associated with the 155-kDa viral protein, it was expressed in Escherichia coli BL21(DE3) cells with thioredoxin, hexahistidine, and S · Tag fused consecutively at its amino terminus, and the fusion protein was purified by metal affinity chromatography. Several RNA fragments, prepared by in vitro transcription, were tested as substrates for the RNA-dependent RNA polymerase (RdRp) activity. Among them, the expressed fusion enzyme was able to generate a ³² P-labeled RNA product when 3′-end RNA fragments of the positive strand or negative strand of BaMV were included in the assay mixture. Dot hybridization assay revealed that the reaction products are complementary to their RNA substrates. Taken together, the evidence suggests that the 155-kDa protein encoded by ORF1 of BaMV has an RdRp activity and should be involved in the replication of BaMV. Mutational analyses demonstrate the importance of the GDD motif in the polymerase activity, and deletion studies suggest that the polymerase activity resides in the carboxyl terminus of the 155-kDa viral protein.