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...