Identification and Characterization of the Escherichia coli -Expressed RNA-Dependent RNA Polymerase of Bamboo Mosaic Virus

Abstract

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 32P-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.

Sequences at the 3′ Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase

“…It is unknown whether the full-length form of this polypeptide, containing the N-terminal and middle portions of the methyltransferase and helicase domains, respectively, would show the same RNA binding. However, since ⌬893 RdRp was demonstrated to be competent for polymerization activity (16) and the interaction with the 3Ј UTR was specific, the interference of the other two domains would be expected to be low. Besides, the hexamer motif protected by RdRp in the footprinting analysis has been observed to play an important role in the replication of BaMV RNA in protoplasts.…”

“…Purification of the recombinant protein ⌬893 RdRp was done as described previously (16), except that two columns were used for further purification before loading into the Talon metal affinity resin column. In brief, once the cells were pelleted and resuspended in 10 ml of sonication buffer (50 mM Tris-HCl [pH 8.0], 10 mM MgCl 2 , 10 mM KCl, 0.1% Triton X-100, 10% glycerol, 5 mM 2-mercaptoethanol), sonication and centrifigation were used to clarify the extract.…”

“…Open reading frame 1 (ORF1), encoding a 155-kDa polypeptide, can be translated directly from the virion RNA in an in vitro rabbit reticulocyte lysate (18). This polypeptide comprises three domains, a methyltransferase-like domain (24), an RNA helicase-like domain (12,13), and an RNA-dependent RNA polymerase domain (1,15,16), from the N to the C terminus. The full-length 155-kDa polypeptide and the C-terminal 80-kDa fragment (⌬893) containing the RdRp domain were overexpressed in Escherichia coli and demonstrated to have RNA-dependent RNA polymerase activities (16).…”

Mutational analysis of a helicase motif-based RNA 5′-triphosphatase/NTPase from bamboo mosaic virus

“…With the RNA 5′triphosphatase and the aforementioned capping enzyme activities, a cap structure could be formed at the 5′ end of BaMV mRNA in vitro. The recombinant RdRp domain showed a template-dependent RNA polymerase activity and had preferential binding activity to the 3′ noncoding region of the viral RNA (Huang et al, 2001;Li et al, 1998).…”

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

“…The preference of 3' positive and 3' negative strand may depend on the specific recognition of RdRp of the special structure of RNA substrate for the initiation of RNA synthesis in a primer dependent manner. The importance of the 3' untranslated region of RNA in the initiation of replication process has been demonstrated in several viruses including bamboo mosaic virus (Li et al, 1998), turnip crinkle virus (Rajendran et al, 2002), picorna virus (Kok and McMinn, 2009) and rotavirus (Chen et al, 2001).…”