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

Huang, Cheng‐Yen; Huang, Yih‐Leh; Meng, Menghsiao; Hsu, Yau‐Heiu; Tsai, Ching‐Hsiu

doi:10.1128/jvi.75.6.2818-2824.2001

Cited by 43 publications

(39 citation statements)

References 31 publications

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“…The importance of these structures in the BaMV replication has been demonstrated in vivo and in vitro (30,33–35). The D domain and the pseudoknot were shown to strongly interact with a recombinant viral RdRp (36). In addition, it has been reported that synthesis of the minus-strand RNA initiates most frequently at the pseudoknot of the 3′-UTR, more specifically at the poly(A) residues of the pseudoknot (37).…”

Section: Introductionmentioning

confidence: 99%

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

Lin

Ding

Hsu

et al. 2006

Nucleic Acids Research

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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.

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Section: Introductionmentioning

confidence: 99%

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

Lin

Ding

Hsu

et al. 2006

Nucleic Acids Research

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“…These structures were demonstrated to be important for minus-strand RNA synthesis in vitro by use of a partially purified replicase from infected Nicotiana benthamiana (4). The interactions of the 3Ј UTR of BaMV RNA with the Escherichia coli-expressed viral RdRp core domain have been demonstrated by electrophoretic mobility shift assay and footprinting analysis (15). Mutants with substitutions, deletions, and insertions introduced into stemloop D failed to replicate efficiently in vivo, indicating that maintaining the integrity of the stem-loop structure is important for BaMV RNA replication (3).…”

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confidence: 99%

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

Lin

Chiu

Chen

et al. 2005

J Virol

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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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“…Although we did not detect binding of the isolated TMV-L RdRp domain to the TMV-L 3Ј-TR in our UV cross-linking assay, binding of the isolated 80-kDa RdRp domain of the bamboo mosaic virus (BaMV) 155-kDa replication protein to the BaMV 3Ј untranslated region was detected using a bandshift assay (11). The BaMV 155-kDa replication protein is analogous to the TMV 183-kDa protein and contains methyltransferase and helicase domains as well as the C-terminal RdRp domain.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Region of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replication Proteins Which Binds Specifically to the Viral 3′-Terminal tRNA-Like Structure

Osman

Buck

2003

J Virol

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Tobacco mosaic virus (TMV) has a 6.3-kb positive-stranded RNA genome which encodes four proteins (reviewed in references 1, 2, and 6). The 126-and 183-kDa proteins have essential replication functions and are translated from the virus genomic RNA. The 126-kDa protein has an N-terminal methyltransferase and guanylyltransferase or capping domain (14, 16) and a C-terminal helicase domain (10, 14). The 183-kDa protein, which is translated as a result of ribosomal readthrough of a termination codon at the end of the open reading frame for the 126-kDa protein, additionally has a domain typical of RNA-dependent RNA polymerases (RdRps) (14). The 35-kDa cell-to-cell movement protein and the 17.5-kDa coat protein are translated from 3Ј-coterminal subgenomic RNAs.The 3Ј-terminal region (3Ј-TR) of TMV RNA can be folded into a tRNA-like structure and adjacent upstream pseudoknots (7, 31) which have been shown to be important for TMV RNA replication in vivo (3, 28) and negative-strand RNA synthesis in vitro (22,33). Regions which were identified as being important for negative-strand synthesis in vitro included the 3Ј-terminal CA sequence, domains D1 (equivalent to a tRNA acceptor arm), D2 (similar to a tRNA anticodon arm), and D3 (an upstream pseudoknotted region), and a central core region C which connects domains D1, D2, and D3. Domain D2 and the central core region C were shown to be the most important elements for binding of the TMV RdRp complex to the viral 3Ј-TR (22).The TMV RdRp complex, isolated from infected plants, consists of the 126-kDa protein, the 183-kDa protein, and a number of plant proteins (21,29,33). Until now, the protein component of the RdRp complex responsible for the specific binding to the TMV RNA 3Ј-TR was unknown. Here we have used UV cross-linking to show that a region of the 126-and 183-kDa proteins downstream of the core methyltransferase domain is responsible for binding of the RdRp to the RNA 3Ј-TR in vitro. We have also identified two aromatic amino acids in this region of the 126-and 183-kDa proteins that are essential for cross-linking to the RNA 3Ј-TR in vitro and for replication of TMV RNA in tomato protoplasts. MATERIALS AND METHODSPreparation of TMV-L RdRp. Membrane-bound viral RdRp was isolated from tomato plants infected with TMV-L by differential and density gradient centrifugation and then solubilized and purified as described by Osman and Buck (20,21).Preparation of cDNA templates and in vitro transcription. RNA t2 and the mutants described in Table 1 were synthesized by in vitro transcription using T7 RNA polymerase and a DNA template generated by PCR from cDNA clones of TMV-L RNA as described previously (22). RNAs corresponding to the 3Ј-terminal 273 nucleotides (nt) of red clover necrotic mosaic virus RNA2 and nt 2466 to 2738 of the vector LITMUS28 were synthesized by in vitro transcription using T7 RNA polymerase and templates described previously (22). RNA transcripts were purified as described in the Ambion manual and assayed spectrophotometrically.Expression of proteins in ...

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

Cited by 43 publications

References 31 publications

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

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

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

Identification of a Region of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replication Proteins Which Binds Specifically to the Viral 3′-Terminal tRNA-Like Structure

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