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A putative nonstructural protein encoded by a satellite RNA associated with bamboo mosaic potexvirus shares 46% identity with the capsid protein of satellite virus of panicum mosaic sobemovirus. The sequence similarity among satellite plant viruses which have no apparent relationship implies a common origin.
The cymbidium mosaic potexvirus (CyMV) banded inclusions and the odontoglossum ringspot tobamovirus (ORSV) paracrystalline inclusions were studied in flowers and leaves of nine orchid cultivars using the confocal laser scanning microscope. The inclusions varied in length and width and were mostly located adjacent to the cell walls. Some ORSV inclusions fully extended across entire infected cells. We propose that the CyMV banded inclusion was formed from virus aggregates which aligned as periodical stacked layers, appearing as cross bands. The virus aggregates were folded into flexible inclusions, giving rise to various shapes and forms. The ORSV paracrystalline inclusions were observed as needle-like crystals. The confocal laser scanning microscope is an effective tool for the study of the three-dimensional structures of plant virus induced inclusions.
Satellite RNA of bamboo mosaic potexvirus (satBaMV) is a linear RNA molecule which encodes a 20-kDa nonstructural protein. Sequences of seven different satBaMV isolates from bamboo hosts in three genera showed 0.7% to 7.5% base variation which spanned the whole RNA molecule. However, the putative 20-kDa open reading frame was all preserved in these isolates. The phylogenetic relationship based on the nucleotide sequence did not show particular grouping of satBaMV from the host in one genus; neither was the grouping of satBaMV evident by location of sampling. Putative secondary structures of the 3' untranslated regions showed a basic pattern with conserved hexanucleotides (ACCUAA) and polyadenylation signal (AAUAAA) located in the loop regions. Although the satBaMV-encoded 20-kDa protein is a nonstructural protein, its predicted secondary structure contains eight-stranded beta-sheets which may form "jelly-roll" structure similar to that found in capsid protein encoded by satellite virus of panicum mosaic virus.
The TGBp1 protein encoded by the first open reading frame of the triple-gene-block (TGB) of bamboo mosaic potexvirus (BaMV) plays important roles in virus movement; one of them is to shape the viral RNA before its being transported from cell to cell. However, TGBp1 mainly forms cytoplasmic inclusions which are devoid of the RNA-binding activity. With the purified TGBp1 inclusions of BaMV, we analyzed the possibility of dissociation of functional TGBp1 from the inclusions. Our data showed that the cytoplasmic inclusions were able to dissociate continuously functional TGBp1, which possessed ATP-binding, ATPase, and RNA-binding activities. Moreover, the dissociation of TGBp1 was significantly enhanced by the presence of viral RNA and capsid protein (CP), consistent with the finding that TGBp1 are able to form ribonucleoprotein complex with viral RNA and CP. Taken together, these results support the idea that the cytoplasmic TGBp1 inclusions are active pools of TGBp1, upon which viral RNA can be shaped into a transferable form.
Mentha x gracilis 'Variegata', an ornamental mint clone first described about 200 years ago, exhibits virus-like vein banding symptoms. Double-stranded RNA and virion isolations revealed the presence of three viruses in a 'Variegata' plant. Cloning and sequencing disclosed that one of the viruses was a previously unidentified species with similarities to members of the Flexiviridae family, designated as Mint virus X (MVX). The complete nucleotide sequence of the virus was determined. Phylogenetic analysis divulged the close relationship of the virus with lily virus X and strawberry mild yellow edge virus, members of the Potexvirus genus. A reverse transcription-polymerase chain reaction protocol was developed and used for detection of MVX in other 'Variegata' plants. All clones tested, obtained from nurseries around the United States were infected with MVX, making the virus a possible causal agent of the variegated symptoms.
Biologically active cDNA clones of cymbidium mosaic potexvirus (CymMV) were synthesized using a population cloning strategy. Three populations of overlapping RT-PCR products encompassing the entire viral RNA of CymMV were ligated into pBluescriptKS+ with T7 RNA polymerase promoter fused to the 5' extreme of the viral cDNA. Capped-RNA in vitro transcripts were infectious. This is the first report of successful synthesis of biologically active CymMV clones. Unlike the conventional methods, population cloning maximizes the probability of obtaining biologically active cDNA clones.
Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for cell-to-cell movement, while Cowpea chlorotic mottle virus (CCMV, a bromovirus) does not. Using bombardment-mediated transcomplementation assays, we investigated whether the movement functions encoded by these viruses potentiate cell-to-cell movement of movement-defective Tomato mosaic virus (ToMV, a tobamovirus) and Potato virus X (PVX, a potexvirus) mutants in Nicotiana benthamiana. Coexpression of CMV 3a and CP, but neither protein alone, complemented the defective movement of ToMV and PVX. A C-terminal deletion in CMV 3a (3a Delta C33) abolished the requirement of CP in transporting the ToMV genome. The action of 3a Delta C33 was inhibited by coexpression of wild-type 3a. These findings were confirmed in tobacco with ToMV-CMV chimeric viruses. Either BMV 3a or CCMV 3a alone efficiently complemented the movement-defective phenotype of the ToMV mutant. Therefore, every 3a protein examined intrinsically possesses the activity required to act as MP. In transcomplementation of the PVX mutant, the activities of BMV 3a, CCMV 3a, and CMV 3a Delta C33 were very low. The activities of the bromovirus 3a proteins were enhanced by coexpression of the cognate CP but the activity of CMV 3a Delta C33 was not. Based on these results, possible roles of cucumo- and bromovirus CPs in cell-to-cell movement are discussed.
The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/DeltaABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40A/DeltaA, -/DeltaB, and -/DeltaC each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40A/DeltaB and -/DeltaC were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMV/DeltaA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain.
Cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) are the two most prevalent and economically important orchid viruses which have attained a world-wide distribution. We have designed one pair of common primers, which were able to detect, simultaneously, both viruses by a single polymerase chain reaction (PCR). Sequence alignment and primer analyses were employed in primer design. The effect of touch-down PCR, primer annealing temperature, number of thermal cycles, MgCl2 concentration and the degree of primer degeneracy, were investigated and applied to the optimisation of PCR conditions in the detection of both viruses in order to achieve equal amplification efficiencies. This is the first successful attempt at using a single pair of PCR primers to detect two viruses belonging to different taxonomic groups simultaneously.
RNA-dependent RNA polymerases (RdRp) isolated from bamboo mosaic potexvirus (BaMV) and potato virus X infected Nicotiana benthamiana plants and solubilized with the detergent NP-40, generated a full-length genomic and two subgenomic double-stranded RNAs of respective viruses in an in vitro RdRp assay containing endogenous RNA templates. Template-dependent and species-specific RdRp activity could be detected after the removal of endogenous RNA templates. The 3' untranslated regions (UTR) containing a stretch of 40 adenylate residues were shown to be an efficient exogenous RNA template for in vitro RdRp reactions. Solution hybridization and nuclease digestion studies revealed that the products transcribed in vitro were minus-sense. Besides using the 3' UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3' terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis. Promoter studies with BaMV RdRp showed that domain D containing the potexviral hexamer motif of the 3' UTR would be the major contributor of minus-sense RNA synthesis in vitro. On the other hand, the pseudoknot domain containing the poly(A) sequences would be sufficient for minus-sense RNA synthesis.
In order to investigate the possible function of the potexviral conserved hexanucleotide sequence (ACc/uUAA) found in the 3' untranslated region of bamboo mosaic potexvirus (BaMV) genomic RNA, each nucleotide in the hexamer motif was substituted. Transcripts derived from wild-type and mutants with a loop deletion or a single-nucleotide substitution were inoculated into protoplasts. The accumulation levels of viral coat protein and RNAs detected from Western and Northern blots of each inoculation were examined after a 48-h incubation. Our data revealed that the nucleotides at positions 4-6 of the hexamer motif cannot be replaced by other nucleotides; the first position of this hexamer is purine specific, and the second position is restricted to pyrimidine. Substitution at the third position has less effect on viral accumulation in protoplasts. In addition to the results reported previously that the E. coli over-expressed BaMV RNA-dependent RNA polymerase could specifically interact with the hexamer motif, the hexanucleotides were thought to serve as a recognition site of viral replicase and the specificity may be derived from the functional groups of each nucleotide of this hexamer motif.
A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.
The complete nucleotide sequence of Plantago asiatica mosaic virus (P1AMV) genomic RNA has been determined. The 6128 nucleotide sequence contains five open reading frames (ORFs) coding for proteins of M(r) 156K (ORF1), 25K (ORF2), 12K (ORF3), 13K (ORF4) and 22K (ORF5). The sequences of these P1AMV proteins exhibit strong homology to the proteins of the other potexviruses. Phylogenetic trees based on the multiple sequence alignments of three conserved domains in ORF1 product and capsid protein reveal a close relationship of P1AMV to papaya mosaic virus and clover yellow mosaic virus. The P1AMV genomic RNA and a major subgenomic RNA (sgRNA) of 0.9 kb have been detected in infected leaves by Northern blot hybridization. The latter sgRNA is the messenger for virus capsid protein and its 5' terminus has been located 23 nucleotides upstream of the initiator codon of the coat protein gene. The P1AMV virion RNA and RNA transcript resembling the 0.9 kb sgRNA have been translated in vitro giving rise to a single major 170K product and a major 22K product, respectively.
Open reading frame 2 of the bamboo mosaic potexvirus (BaMV) genome encodes a 28 kDa protein, the first of the "triple-gene-block' of BaMV which is believed to play a role in cell-to-cell movement of the virus in host plants. The 28 kDa protein was expressed in Escherichia coli and polyclonal antiserum was raised in a rabbit. Western blot analyses showed that the 28 kDa protein was associated mainly with components in the cell wall and 30000 g pellet fractions of a BaMV-infected leaf homogenate. Immunogold electron microscopy of infected leaf tissues revealed that the 28 kDa protein was associated with electron-dense crystal-line bodies (EDCBs) in the cytoplasm and nuclei. Nuclear EDCBs were found closely associated with nucleoli. Gold-labelled EDCB-like structures were also detected in the cytoplasm, but not within nuclei, in protoplasts up to 48 h post-inoculation. No specific labelling of the 28 kDa protein was found within any cytoplasmic structures or within cell walls.
Purified virions of bamboo mosaic potexvirus-V isolate (BaMV-V) were found to contain three major RNA species, the 6.4 kb genomic RNA and two RNAs of 2.0 and 1.0 kb, in addition to associated satellite RNA (0.85 kb). Results of Northern blot hybridization, primer extension analysis and cDNA sequencing showed that the packaged 2.0 and 1.0 kb RNAs of BaMV-V were subgenomic RNAs. In contrast, in the BaMV-O isolate, only genomic RNA was packaged and encapsidated subgenomic RNAs were not detectable. The transcription initiation sites for the 2.0 and 1.0 kb subgenomic RNAs of BaMV-V were located 1 1 and 16 nt upstream of the initiation codon of open reading frames (ORFs) 2 and 5, respectively. The 2.0 and 1.0 kb subgenomic RNAs functioned as messengers for the ORF2 protein and capsid protein, respectively. Packaging of the 1.0 kb subgenomic RNAs resulted in the formation of rod-shaped particles about 70 nm in length. Our results indicate that BaMV isolates have evolved distinctly for packaging of subgenomic RNAs.
RNA silencing in transgenic and virus-infected plants involves a mobile silencing signal that can move cell-to-cell and systemically through the plant. It is thought that this signal can influence long-distance movement of viruses because protein suppressors of silencing encoded in viral genomes are required for long-distance virus movement. However, until now, it was not known whether the mobile signal could also influence short-range virus movement between cells. Here, through random mutation analysis of the Potato Potexvirus X (PVX) silencing suppressor P25, we provide evidence that it does. All mutants that were defective for silencing suppression were also non-functional in viral cell-to-cell movement. However, we identified mutant P25 proteins that were functional as silencing suppressors but not as movement proteins and we conclude that suppression of silencing is not sufficient to allow virus movement between cells: there must be a second P25 function that is independent of silencing but also required for cell-to-cell movement. Consistent with this hypothesis, we identified two classes of suppressor-inactive P25 mutants. One class of these mutants is proposed to be functional for the accessory function because their failure to support PVX movement could be complemented by heterologous suppressors of silencing. The second class of P25 mutants is considered defective for both the suppressor and second functions because the heterologous silencing suppressors did not restore virus movement. It is possible, based on analyses of short interfering RNA accumulation, that P25 suppresses silencing by interfering with either assembly or function of the effector complexes of RNA silencing.
Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPdeltaN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPdeltaN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPdeltaN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.
RNAs transcribed from a full-length infectious cDNA clone of the bamboo mosaic potexvirus (strain O) genome, pBaMV-O, were infectious to Nicotiana benthamiana plants. Mutant genomes in which the poly(A) tail is absent or replaced by a 3' tRNA-like structure from turnip yellow mosaic virus RNA failed to amplify detectably in N. benthamiana protoplasts. No amplification was detected in protoplasts inoculated with transcripts containing 4, 7, or 10 adenylate residues at the 3' end, whereas transcript inocula with 15 adenylate residues resulted in coat protein accumulation to a level 26% of that resulting from inoculation with transcripts with 25 adenylate residues (designated as wild type). Coat protein accumulation levels of 69 and 98% relative to wild type were observed after inoculation of protoplasts with transcripts bearing poly(A) tails 18 and 22 nucleotides long, respectively. The presence of a putative 3' pseudoknot structure including at least 13 adenylate residues of the 3'-terminal poly(A) tail was supported by enzymatic and chemical structural analysis. The functional relevance of this putative pseudoknot was tested by mutations that affected basepairing within the pseudoknot. These results support the existence of functional 3' pseudoknot that includes part of the 3' poly(A) tail.