Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection.In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation.The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3′ untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis.The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis.
Bamboo mosaic virus (BaMV); glutathione (GSH); glutathione S-transferase (GST); in vitro RNA replication; redox; viral RNA replication; virus-induced gene silencing (VIGS)
Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses.
Bamboo 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.
To 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.
Among 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.
Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.
The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3′ untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of ∼35 and ∼55 kDa were specifically cross-linked with RNA elements, whereupon the ∼35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3′ UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3′ UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication.
Satellite RNAs associated with Bamboo mosaic virus (satBaMVs) depend on BaMV for replication and encapsidation. Certain satBaMVs isolated from natural fields significantly interfere with BaMV replication. The 5′ apical hairpin stem loop (AHSL) of satBaMV is the major determinant in interference with BaMV replication. In this study, by in vivo competition assay, we revealed that the sequence and structure of AHSL, along with specific nucleotides (C60 and C83) required for interference with BaMV replication, are also involved in replication competition among satBaMV variants. Moreover, all of the 5′ ends of natural BaMV isolates contain the similar AHSLs having conserved nucleotides (C64 and C86) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C86 was essential for BaMV replication, and that replacement of C64 with U reduced replication efficiency. The non-interfering satBaMV interfered with BaMV replication with the BaMV-C64U mutant as helper. These findings suggest that two cytosines at the equivalent positions in the AHSLs of BaMV and satBaMV play a crucial role in replication competence. The downregulation level, which is dependent upon the molar ratio of interfering satBaMV to BaMV, implies that there is competition for limited replication machinery.
Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta.
The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP).
Following inoculation with BaMV, N. benthamiana displayed differential gene expression in response to the infection. Isolation, cloning, and sequencing analysis using cDNA-AFLP furnished 90 cDNA fragments with eight pairs of selective primers. Fifteen randomly selected genes were used for a combined virus-induced gene silencing (VIGS) knockdown experiment, using BaMV infection to investigate the roles played by these genes during viral infection, specifically addressing the means by which these genes influence the accumulation of BaMV protein. Nine of the 15 genes showed either a positive or a negative influence on the accumulation of BaMV protein. Six knockdown plants showed an increase in the accumulation of BaMV, suggesting that they played a role in the resistance to viral infection, while three plants showed a reduction in coat protein, indicating a positive influence on the accumulation of BaMV in plants. An interesting observation was that eight of the nine plants showing an increase in BaMV coat protein were associated with cell rescue, defense, death, aging, signal transduction, and energy production.
This study reports an efficient and straightforward method for the identification of host genes involved in viral infection. We succeeded in establishing a cDNA-AFLP system to help track changes in gene expression patterns in N. benthamiana plants when infected with BaMV. The combination of both DNA-AFLP and VIGS methodologies made it possible to screen a large number of genes and identify those associated with infections of plant viruses. In this report, 9 of the 15 analyzed genes exhibited either a positive or a negative influence on the accumulation of BaMV in N. benthamiana plants.
Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana.
Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (−) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.
The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.
Bamboo mosaic virus (BaMV) is a 6.4-kb positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The 155-kDa viral replicase, the product of ORF1, comprises an N-terminal S-adenosyl-l-methionine (AdoMet)-dependent guanylyltransferase, a nucleoside triphosphatase/RNA 5′-triphosphatase, and a C-terminal RNA-dependent RNA polymerase (RdRp). To search for cellular factors potentially involved in the regulation of replication and/or transcription of BaMV, the viral RdRp domain was targeted as bait to screen against a leaf cDNA library of Nicotiana benthamiana using a yeast two-hybrid system. A putative methyltransferase (PNbMTS1) of 617 amino acid residues without an established physiological function was identified. Cotransfection of N. benthamiana protoplasts with a BaMV infectious clone and the PNbMTS1-expressing plasmid showed a PNbMTS1 dosage-dependent inhibitory effect on the accumulation of BaMV coat protein. Deletion of the N-terminal 36 amino acids, deletion of a predicted signal peptide or transmembrane segment, or mutations in the putative AdoMet-binding motifs of PNbMTS1 abolished the inhibitory effect. In contrast, suppression of PNbMTS1 by virus-induced gene silencing in N. benthamiana increased accumulation of the viral coat protein as well as the viral genomic RNA. Collectively, PNbMTS1 may function as an innate defense protein against the accumulation of BaMV through an uncharacterized mechanism.
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.
satellite RNA; satellite virus; replication; RNA silencing; pathogenicity
The triple-gene-block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which was proposed to be involved in viral RNA binding during virus transport. Here, we report on the RNA-binding properties of TGBp2. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates intracellular delivery of viral RNA through non-specific protein-RNA interaction.
The satellite RNA of bamboo mosaic virus (satBaMV) has a single open reading frame encoding a non-structural protein, P20, which facilitates long-distance movement of satBaMV in BaMV and satBaMV co-infected plants. Immunohistochemistry and immunoelectron microscopy revealed that the P20 protein accumulated in the cytoplasm and nuclei in co-infected cells. P20 and the helper virus coat protein (CP) were highly similar in their subcellular localization, except that aggregates of BaMV virions were not labelled with anti-P20 serum. The BaMV CP protein was fairly abundant in mesophyll cells, whilst P20 was more frequently detected in mesophyll cells and vascular tissues. The expression kinetics of the P20 protein was similar to but slightly earlier than that of CP in co-infected Bambusa oldhamii protoplasts and Nicotiana benthamiana leaves. However, satBaMV-encoded protein levels declined rapidly in the late phase of co-infection. During co-infection, in addition to the intact P20, a low-molecular-mass polypeptide of 16 kDa was identified as a P20 C-terminally truncated product; the possible method of generation of the truncated protein is discussed.
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.
Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5′ untranslated region (UTR) of BSL6. The 5′ UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5′ UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5′ UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.
Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome with a 5′-cap structure and a 3′ poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3′ untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3′ UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.
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/Δ5, 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/Δ16 and Ba-77/Δ31, 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.
Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5′ cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an ∼10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m7GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m7GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m7GTP sustained the formation of the m7GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m7GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m7GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m7GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m7GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.
The 3′ terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5′ portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3′ untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5′ terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5′ rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5′-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from Nicotiana benthamiana protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5′ RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3′ UTR of BaMV genomic RNA.
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 γ 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 m7GpppG 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.
The 3′ untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555–565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that the Escherichia coli-expressed RNA-dependent RNA polymerase domain (Δ893) derived from BaMV open reading frame 1 could specifically bind to the 3′ UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3′ UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3′ UTR showed that Δ893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Δ893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.
Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5′ end of the viral genome. To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in Saccharomyces cerevisiae. All three versions of BaMV ORF1 proteins could be radiolabeled by [α-32P]GTP, which is a characteristic of guanylyltransferase activity. The presence of S-adenosylmethionine (AdoMet) was essential for this enzymatic activity. Thin-layer chromatography analysis suggests that the radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 protein is m7GMP. The N-terminal domain also exhibited methyltransferase activity that catalyzes the transfer of the [3H]methyl group from AdoMet to GTP or guanylylimidodiphosphate. Therefore, during cap structure formation in BaMV, methylation of GTP may occur prior to transguanylation as for alphaviruses and brome mosaic virus. This study establishes the association of RNA capping activity with the N-terminal domain of the replicase of potexviruses and further supports the idea that the reaction sequence of RNA capping is conserved throughout the alphavirus-like superfamily of RNA viruses.
Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded positive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full-length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter-like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protein (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium quinoa when coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by deletion mutation, we concluded that an SGP contains one core promoterlike sequence (nt −30 through +16), two upstream enhancers (nt −59 through −31 and −91 through −60), and one downstream enhancer (nt +17 through +52), when the transcription initiation site is taken as +1. Site-directed mutagenesis and compensatory mutation to disrupt and restore potential base pairing in the core promoter-like sequence suggest that the stem-loop structure is important for the function of SGP in vivo. Likewise, the insertion of a putative SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.
A satellite RNA of 836 nucleotides [excluding the poly(A) tail] depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsidation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein (P20). The P20 protein with eight histidine residues at the C terminus was overexpressed in Escherichia coli. Experiments of gel retardation, UV cross-linking, and Northwestern hybridization demonstrated that purified P20 was a nucleic-acid-binding protein. The binding of P20 to nucleic acids was strong and highly cooperative. P20 preferred binding to satBaMV- or BaMV-related sequences rather than to nonrelated sequences. By deletion analysis, the P20 binding sites were mainly located at the 5′ and 3′ untranslated regions of satBaMV RNA, and the RNA-protein interactions could compete with the poly(G) and, less efficiently, with the poly(U) homopolymers. The N-terminal arginine-rich motif of P20 was the RNA binding domain, as shown by in-frame deletion analysis. This is the first report that a plant virus satellite RNA-encoded nonstructural protein preferentially binds with nucleic acids.