The tertiary structure in the 3′-untranslated region (3′-UTR) of Bamboo mosaic virus (BaMV) RNA is known to be involved in minus-strand RNA synthesis. Proteins found in the RNA-dependent RNA polymerase (RdRp) fraction of BaMV-infected leaves interact with the radio labeled 3′-UTR probe in electrophoretic mobility shift assays (EMSA). Results derived from the ultraviolet (UV) cross-linking competition assays suggested that two cellular factors, p43 and p51, interact specifically with the 3′-UTR of BaMV RNA. p43 and p51 associate with the poly(A) tail and the pseudoknot of the BaMV 3′-UTR, respectively. p51-containing extracts specifically down-regulated minus-strand RNA synthesis when added to in vitro RdRp assays. LC/MS/MS sequencing indicates that p43 is a chloroplast phosphoglycerate kinase (PGK). When the chloroplast PKG levels were knocked down in plants, using virus-induced gene silencing system, the accumulation level of BaMV coat protein was also reduced.

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3 terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/⌬5, with a deletion of the 3-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/⌬16 and Ba-77/⌬31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3 end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.There are at least two major steps involved in the replication of positive-sense viral RNA: the first is the synthesis of the complementary strand of genomic RNA, which is followed by the generation of progeny RNAs using the newly synthesized minus-strand RNA templates (37). For these reactions to proceed faithfully, the 3Ј ends of both plus-and minus-strand RNAs must contain important elements, the sequences and/or structures needed to direct the replicase to initiate the RNA synthesis (7, 18). The 3Ј-terminal secondary structures of Alfalfa mosaic virus RNAs (12,13,33), Turnip crinkle virus RNAs (39), and Tomato bush stunt virus defective interfering RNA (8) and the 3Ј tRNA-like structures (TLS) of Brome mosaic virus (BMV), Tobacco mosaic virus, and Turnip yellow mosaic virus (TYMV) RNAs have been identified to be important for minus-strand RNA synthesis (5,6,17,26,36).Bamboo mosaic virus (BaMV) is a single-stranded positivesense RNA virus. Its 6,366-nucleotide (nt) genome [excluding the poly(A) tail] contains a 5Ј m 7 GpppG structure and a 3Ј poly(A) tail (24). Five open reading frames (ORF) encoding polypeptides of 155, 28, 13, 6, and 25 kDa could be identified in the genome. Besides the genomic RNA which encodes the 155-kDa protein comprising a capping enzyme domain (21, 22), a 5Ј triphosphatase and helicase-like domain (9,11,16), and an RNA-dependent RNA polymerase (RdRp) activi...

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A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.

BackgroundBamboo mosaic virus (BaMV) and the Potato virus X (PVX) are members of the genus Potexvirus and have a single-stranded positive-sense RNA genome. The 3′-untranslated region (UTR) of the BaMV RNA genome was mapped structurally into ABC (a cloverleaf-like), D (a stem-loop), and E (pseudoknot) domains. The BaMV replicase complex that was isolated from the infected plants was able to recognize the 3′ UTR of PVX RNA to initiate minus-strand RNA synthesis in vitro.ResultsTo investigate whether the 3′ UTR of PVX RNA is also compatible with BaMV replicase in vivo, we constructed chimera mutants using a BaMV backbone containing the PVX 3′ UTR, which was inserted in or used to replace the various domains in the 3′ UTR of BaMV. None of the mutants, except for the mutant with the PVX 3′ UTR inserted upstream of the BaMV 3′ UTR, exhibited a detectable accumulation of viral RNA in Nicotiana benthamiana plants. The in vitro BaMV RdRp replication assay demonstrated that the RNA products were generated by the short RNA transcripts, which were derived from the chimera mutants to various extents. Furthermore, the Vmax/KM of the BaMV 3′ UTR (rABCDE) was approximately three fold higher than rABCP, rP, and rDE in minus-strand RNA synthesis. These mutants failed to accumulate viral products in protoplasts and plants, but were adequately replicated in vitro.ConclusionsAmong the various studied BaMV/PVX chimera mutants, the BaMV-S/PABCDE that contained non-interrupted BaMV 3′ UTR was the only mutant that exhibited a wild-type level of viral product accumulation in protoplasts and plants. These results indicate that the continuity of the domains in the 3′ UTR of BaMV RNA was not interrupted and the domains were not replaced with the 3′ UTR of PVX RNA in vivo.