IDENTIFICATION ACID CHARACTERIZATION OF THE ESCHERICHIA COLI-EXPRESSED RNA-DEPENDENT RNA-POLYMERASE OF BAMBOO MOSAIC-VIRUS

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 invol ved in the replication and the formation of cap structure at the 5' en d of the viral genome. To characterize the activities associated with the 155-kDa viral protein, it was expressed in Escherichia coli BL21(D E3) cells with thioredoxin, hexahistidine, and S Tag fused consecutive ly 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 pol ymerase (RdRp) activity. Among them, the expressed fusion enzyme was a ble to generate a P-32-labeled RNA product when 3'-end RNA fragments o f the positive strand or negative strand of BaMV were included in the assay mixture. Dot hybridization assay revealed that the reaction prod ucts are complementary to their RNA substrates. Taken together, the ev idence suggests that the 155-kDa protein encoded by ORF1 of BaMV has a n RdRp activity and should be involved in the replication of BaMV, Mut ational analyses demonstrate the importance of the GDD motif in the po lymerase activity, and deletion studies suggest that the polymerase ac tivity resides in the carboxyl terminus of the 155-kDa viral protein.