MATERIALS AND METHODSVirus Isolates. BaMV isolate V (BaMV-V) contains satBaMV (14). BaMV-L is an isolate derived from BaMV-V and free of satBaMV (designated as

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The Bamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus. Previously, we identified that the chloroplast phosphoglycerate kinase (chl-PGK) from Nicotiana benthamiana is one of the viral RNA binding proteins involved in the BaMV infection cycle. Because chl-PGK is transported to the chloroplast, we hypothesized that chl-PGK might be involved in viral RNA localization in the chloroplasts. To test this hypothesis, we constructed two green fluorescent protein (GFP)-fused mislocalized PGK mutants, the transit peptide deletion mutant (NO TRANSIT PEPTIDE [NOTP]-PGK-GFP) and the nucleus location mutant (nuclear location signal [NLS]-PGK-GFP). Using confocal microscopy, we demonstrated that NOTP-PGK-GFP and NLS-PGK-GFP are localized in the cytoplasm and nucleus, respectively, in N. benthamiana plants. When NOTP-PGK-GFP and NLS-PGK-GFP are transiently expressed, we observed a reduction in BaMV coat protein accumulation to 47% and 27% that of the wild-type PGK-GFP, respectively. To localize viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence. Using confocal microscopy, we observed that BaMV viral RNA localizes to chloroplasts. Furthermore, elongation factor1a fused with the transit peptide derived from chl-PGK or with a Rubisco small subunit can partially restore BaMV accumulation in NbPGK1-knockdown plants by helping BaMV target chloroplasts.

The view that satellite RNAs (satRNAs) and satellite viruses are purely molecular parasites of their cognate helper viruses has changed. The molecular mechanisms underlying the synergistic and/or antagonistic interactions among satRNAs/satellite viruses, helper viruses, and host plants are beginning to be comprehended. This review aims to summarize the recent achievements in basic and practical research, with special emphasis on the involvement of RNA silencing mechanisms in the pathogenicity, population dynamics, and, possibly, the origin(s) of these subviral agents. With further research following current trends, the comprehensive understanding of satRNAs and satellite viruses could lead to new insights into the trilateral interactions among host plants, viruses, and satellites.

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.

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...

The protein encoded by the first gene of the triple gene block (TGBp1) of potexviruses is required for movement of the viruses. It has been reported that single ArgRAla substitutions at position 11, 16 or 21 of TGBp1 of Bamboo mosaic virus (BaMV) eliminate its RNA-binding activity, while substitutions at position 16 or 21 only affect its NTPase activity (Liou et al., Virology 277, 336-344, 2000). However, it remains unclear whether these ArgRAla substitutions also affect the movement of BaMV in plants. To address this question, six mutants of BaMV, each containing either a single-or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed and used to infect Chenopodium quinoa and Nicotiana benthamiana. We found that all of the BaMV mutants were able to replicate in protoplasts of N. benthamiana. However, only the mutant with an Arg-11RAla substitution in TGBp1 remained capable of movement from cell to cell in plants. Mutants with Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 replaced with alanine were defective in virus movement. This defect was suppressed when a wild-type TGBp1 allele was co-introduced into the cells using a novel satellite replicon. The ability to trans-complement the movement defect by the wild-type TGBp1 strongly suggests that the ArgRAla substitution at position 16 or 21 of TGBp1, which diminishes the RNA-binding and NTPase activities of TGBp1, also eliminates the capability of BaMV to move from cell to cell in host plants.