Ching-Hsiu Tsai

Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus

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.

Sequences at the 3′ Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase

ABSTRACT 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 theEscherichia 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.

The Synthesis of Minus-Strand RNA of Bamboo Mosaic Potexvirus Initiates from Multiple Sites within the Poly(A) Tail

ABSTRACT 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.

Chloroplast Phosphoglycerate Kinase Is Involved in the Targeting of Bamboo mosaic virus to Chloroplasts in Nicotiana benthamiana Plants

The BaMV RNA binds to chloroplast phosphoglycerate kinase and is targeted to chloroplast for replication. 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.

Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism

BackgroundThe 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).ResultsFollowing 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.ConclusionsThis 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.

The AAUAAA Motif of Bamboo Mosaic Virus RNA Is Involved in Minus-Strand RNA Synthesis and Plus-Strand RNA Polyadenylation

ABSTRACT 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.

Suppression of Bamboo Mosaic Virus Accumulation by a Putative Methyltransferase in Nicotiana benthamiana

ABSTRACT 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.

Phosphorylation of Coat Protein by Protein Kinase CK2 Regulates Cell-to-Cell Movement of Bamboo mosaic virus Through Modulating RNA Binding

In this study, we investigated the fine regulation of cell-to-cell movement of Bamboo mosaic virus (BaMV). We report that the coat protein (CP) of BaMV is phosphorylated in planta at position serine 241 (S241), in a process involving Nicotiana benthamiana casein kinase 2α (NbCK2α). BaMV CP and NbCK2α colocalize at the plasmodesmata, suggesting that phosphorylation of BaMV may be involved in its movement. S241 was mutated to examine the effects of temporal and spatial dysregulation of phosphorylation on i) the interactions between CP and viral RNA and ii) the regulation of cell-to-cell movement. Replacement of S241 with alanine did not affect RNA binding affinity but moderately impaired cell-to-cell movement. A negative charge at position 241 reduced the ability of CP to bind RNA and severely interfered with cell-to-cell movement. Deletion of residues 240 to 242 increased the affinity of CP to viral RNA and dramatically impaired cell-to-cell movement. A threonine at position 241 changed the binding preference of CP toward genomic RNA and inhibited cell-to-cell movement. Together, these results reveal a fine regulatory mechanism for the cell-to-cell movement of BaMV, which involves the modulation of RNA binding affinity through appropriate phosphorylation of CP by NbCK2α.

Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

ABSTRACT 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.

The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus

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.