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1
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Lin KY, Huang YW, Hou LY, Chen HC, Wu Y, Chen IH, Huang YP, Lee SC, Hu CC, Tsai CH, Hsu YH, Lin NS. Proviral insights of glycolytic enolase in Bamboo mosaic virus replication associated with chloroplasts and mitochondria. Proc Natl Acad Sci U S A 2025; 122:e2415089122. [PMID: 40327700 PMCID: PMC12088382 DOI: 10.1073/pnas.2415089122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2024] [Accepted: 03/20/2025] [Indexed: 05/08/2025] Open
Abstract
Diverse single-stranded RNA viruses employ different host cellular organelles or membrane systems to compartmentalize their replication intermediates and proviral factors, ensuring robust replication. Replication of Bamboo mosaic virus (BaMV), an Alphaflexiviridae family, is tightly associated with chloroplasts and dynamic cytosolic viral replication complex (VRC) clusters. BaMV VRC clusters comprise double-stranded viral RNA, BaMV replicase (RepBaMV), and mitochondrial outer membrane protein, voltage-dependent anion channel (VDAC). In this study, we demonstrate that host glycolytic enolase (ENO) binds to untranslated regions of BaMV RNA independently of ENO hydrolytic activity. However, the structural integrity of ENO is essential for its direct interaction with RepBaMV, and its positive regulating role in BaMV replication and the size of BaMV VRC clusters. Additionally, ENO, pyruvate kinase (PYK), and VDAC colocalize within cytosolic BaMV VRC clusters embedded in the convoluted endomembrane reticulum (ER) along with ER-targeted viral movement proteins under BaMV infection. This association suggests that the ENO-PYK-VDAC metabolon, with ENO serving as a scaffold to link chloroplasts and mitochondria, may play a pivotal role in BaMV robust replication. Collectively, our findings offer significant insights into how glycolytic ENO acts in BaMV replication.
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Affiliation(s)
- Kuan-Yu Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei11529, Taiwan
| | - Ying-Wen Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
- Advanced Plant and Food Crop Biotechnology Center, National Chung Hsing University, Taichung40227, Taiwan
| | - Liang-Yu Hou
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei11529, Taiwan
| | - Hsin-Chuan Chen
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei11529, Taiwan
| | - Yu Wu
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei11529, Taiwan
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
- Advanced Plant and Food Crop Biotechnology Center, National Chung Hsing University, Taichung40227, Taiwan
| | - Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
- Advanced Plant and Food Crop Biotechnology Center, National Chung Hsing University, Taichung40227, Taiwan
| | - Shu-Chuan Lee
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei11529, Taiwan
| | - Chung-Chi Hu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
- Advanced Plant and Food Crop Biotechnology Center, National Chung Hsing University, Taichung40227, Taiwan
| | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei11529, Taiwan
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung40227, Taiwan
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2
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Úbeda JR, Aranda MA, Donaire L. Alphaflexiviridae in Focus: Genomic Signatures, Conserved Elements and Viral-Driven Cellular Remodeling. Viruses 2025; 17:611. [PMID: 40431623 PMCID: PMC12115993 DOI: 10.3390/v17050611] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2025] [Revised: 04/15/2025] [Accepted: 04/21/2025] [Indexed: 05/29/2025] Open
Abstract
The family Alphaflexiviridae comprises plant- and fungus-infecting viruses with single-stranded, positive-sense RNA genomes ranging from 5.4 to 9 kb. Their virions are flexuous and filamentous, measuring 470-800 nm in length and 12-13 nm in diameter. The family includes 72 recognized species, classified into six genera: Allexivirus, Lolavirus, Platypuvirus, Potexvirus (plant-infecting), and Botrexvirus and Sclerodarnavirus (fungus-infecting). The genus Potexvirus is the largest, with 52 species, including Potexvirus ecspotati (potato virus X), an important crop pathogen and plant virology model. The genera are distinguished by genome organization and host range, while species differentiation relies on nucleotide and protein sequence identity thresholds. In this review, we summarize the current knowledge on the genomic structure, conserved genes, and phylogenetic relationships within Alphaflexiviridae, with a particular focus on the replicase and coat protein genes as signature markers. Additionally, we update the model of cellular remodeling driven by the triple gene block proteins, which are essential for virus movement, among other viral functions. Beyond their biological significance, alphaflexiviruses serve as valuable models for studying virus-host dynamics and hold potential applications in plant disease control and biotechnology. This review provides an updated framework for understanding Alphaflexiviridae and their broader impact on plant virology.
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Affiliation(s)
| | | | - Livia Donaire
- Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, Department of Stress Biology and Plant Pathology, P.O. Box 164, Espinardo, 30100 Murcia, Spain; (J.R.Ú.); (M.A.A.)
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3
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Huang Y, Chen I, Kao Y, Hsu Y, Tsai C. The gibberellic acid derived from the plastidial MEP pathway is involved in the accumulation of Bamboo mosaic virus. THE NEW PHYTOLOGIST 2022; 235:1543-1557. [PMID: 35524450 PMCID: PMC9543464 DOI: 10.1111/nph.18210] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Accepted: 04/12/2022] [Indexed: 06/14/2023]
Abstract
A gene upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) infection was revealed as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (NbDXR). DXR is the key enzyme in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway that catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol-4-phosphate. Knockdown and overexpression of NbDXR followed by BaMV inoculation revealed that NbDXR is involved in BaMV accumulation. Treating leaves with fosmidomycin, an inhibitor of DXR function, reduced BaMV accumulation. Subcellular localization confirmed that DXR is a chloroplast-localized protein by confocal microscopy. Furthermore, knockdown of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, one of the enzymes in the MEP pathway, also reduced BaMV accumulation. The accumulation of BaMV increased significantly in protoplasts treated with isopentenyl pyrophosphate. Thus, the metabolites of the MEP pathway could be involved in BaMV infection. To identify the critical components involved in BaMV accumulation, we knocked down the crucial enzyme of isoprenoid synthesis, NbGGPPS11 or NbGGPPS2. Only NbGGPPS2 was involved in BaMV infection. The geranylgeranyl pyrophosphate (GGPP) synthesized by NbGGPPS2 is known for gibberellin synthesis. We confirmed this result by supplying gibberellic acid exogenously on leaves, which increased BaMV accumulation. The de novo synthesis of gibberellic acid could assist BaMV accumulation.
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Affiliation(s)
- Ying‐Ping Huang
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - I‐Hsuan Chen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Yu‐Shun Kao
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Yau‐Heiu Hsu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
- Advaced Plant Biotechnology CenterNational Chung Hsing UniversityTaichung402Taiwan
| | - Ching‐Hsiu Tsai
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
- Advaced Plant Biotechnology CenterNational Chung Hsing UniversityTaichung402Taiwan
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4
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Chen I, Chen X, Chiu G, Huang Y, Hsu Y, Tsai C. The function of chloroplast ferredoxin-NADP + oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana. MOLECULAR PLANT PATHOLOGY 2022; 23:503-515. [PMID: 34918877 PMCID: PMC8916203 DOI: 10.1111/mpp.13174] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 11/17/2021] [Accepted: 11/21/2021] [Indexed: 05/08/2023]
Abstract
A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP+ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP+ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.
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Affiliation(s)
- I‐Hsuan Chen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan
| | - Xiang‐Yu Chen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan
| | - Guan‐Zhi Chiu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan
| | - Ying‐Ping Huang
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan
| | - Yau‐Heiu Hsu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan
- Advaced Plant Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan
| | - Ching‐Hsiu Tsai
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan
- Advaced Plant Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan
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5
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Huang YP, Hou PY, Chen IH, Hsu YH, Tsai CH, Cheng CP. Dissecting the role of a plant-specific Rab5 small GTPase NbRabF1 in Bamboo mosaic virus infection. JOURNAL OF EXPERIMENTAL BOTANY 2020; 71:6932-6944. [PMID: 32926136 DOI: 10.1093/jxb/eraa422] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
NbRabF1, a small GTPase from Nicotiana benthamiana and a homolog of Arabidopsis thaliana Ara6, plays a key role in regulating Bamboo mosaic virus (BaMV) movement by vesicle transport between endosomal membranes. Reducing the expression of NbRabF1 in N. benthamiana by virus-induced gene silencing decreased the accumulation of BaMV, and with smaller infection foci on inoculated leaves, but had no effect in protoplasts. Furthermore, transient expression of NbRabF1 increased the accumulation of BaMV in inoculated leaves. Thus, NbRabF1 may be involved in the cell-to-cell movement of BaMV. The potential acyl modification sites at the second and third amino acid positions of NbRabF1 were crucial for membrane targeting and BaMV accumulation. The localization of mutant forms of NbRabF1 with the GDP-bound (donor site) and GTP-bound (acceptor site) suggested that NbRabF1 might regulate vesicle trafficking between the Golgi apparatus and plasma membrane. Furthermore, GTPase activity could also be involved in BaMV cell-to-cell movement. Overall, in this study, we identified a small GTPase, NbRabF1, from N. benthamiana that interacts with its activation protein NbRabGAP1 and regulates vesicle transport from the Golgi apparatus to the plasma membrane. We suggest that the BaMV movement complex might move from cell to cell through this vesicle trafficking route.
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Affiliation(s)
- Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Pei-Yu Hou
- Department of Life Sciences, Tzu Chi University, Hualien, Taiwan
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Yau-Huei Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Advanced Plant Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Advanced Plant Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
- Research Center for Sustainable Energy and Nanotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Chi-Ping Cheng
- Department of Life Sciences, Tzu Chi University, Hualien, Taiwan
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6
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Alazem M, He MH, Chang CH, Cheng N, Lin NS. Disrupting the Homeostasis of High Mobility Group Protein Promotes the Systemic Movement of Bamboo mosaic virus. FRONTIERS IN PLANT SCIENCE 2020; 11:597665. [PMID: 33424893 PMCID: PMC7793662 DOI: 10.3389/fpls.2020.597665] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Accepted: 11/11/2020] [Indexed: 05/21/2023]
Abstract
Viruses hijack various organelles and machineries for their replication and movement. Ever more lines of evidence indicate that specific nuclear factors are involved in systemic trafficking of several viruses. However, how such factors regulate viral systemic movement remains unclear. Here, we identify a novel role for Nicotiana benthamiana high mobility group nucleoprotein (NbHMG1/2a) in virus movement. Although infection of N. benthamiana with Bamboo mosaic virus (BaMV) decreased NbHMG1/2a expression levels, nuclear-localized NbHMG1/2a protein was shuttled out of the nucleus into cytoplasm upon BaMV infection. NbHMG1/2a knockdown or even overexpression did not affect BaMV accumulation in inoculated leaves, but it did enhance systemic movement of the virus. Interestingly, the positive regulator Rap-GTPase activation protein 1 was highly upregulated upon infection with BaMV, whereas the negative regulator thioredoxin h protein was greatly reduced, no matter if NbHMG1a/2a was silenced or overexpressed. Our findings indicate that NbHMG1/2a may have a role in plant defense responses. Once its homeostasis is disrupted, expression of relevant host factors may be perturbed that, in turn, facilitates BaMV systemic movement.
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Affiliation(s)
| | | | | | | | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
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7
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Chiu LY, Chen IH, Hsu YH, Tsai CH. The Lipid Transfer Protein 1 from Nicotiana benthamiana Assists Bamboo mosaic virus Accumulation. Viruses 2020; 12:E1361. [PMID: 33261222 PMCID: PMC7760991 DOI: 10.3390/v12121361] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2020] [Revised: 11/23/2020] [Accepted: 11/25/2020] [Indexed: 12/16/2022] Open
Abstract
Host factors play a pivotal role in regulating virus infection. Uncovering the mechanism of how host factors are involved in virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo mosaic virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.
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Affiliation(s)
- Ling-Ying Chiu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan; (L.-Y.C.); (I.-H.C.); (Y.-H.H.)
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan; (L.-Y.C.); (I.-H.C.); (Y.-H.H.)
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan; (L.-Y.C.); (I.-H.C.); (Y.-H.H.)
- Advanced Plant Biotechnology Center, National Chung Hing University, Taichung 402, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan; (L.-Y.C.); (I.-H.C.); (Y.-H.H.)
- Advanced Plant Biotechnology Center, National Chung Hing University, Taichung 402, Taiwan
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Mudgal R, Mahajan S, Tomar S. Inhibition of Chikungunya virus by an adenosine analog targeting the SAM-dependent nsP1 methyltransferase. FEBS Lett 2019; 594:678-694. [PMID: 31623018 PMCID: PMC7164056 DOI: 10.1002/1873-3468.13642] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2019] [Revised: 09/23/2019] [Accepted: 10/02/2019] [Indexed: 01/02/2023]
Abstract
Alphaviruses, including Chikungunya (CHIKV) and Venezuelan equine encephalitis virus (VEEV), are among the leading causes of recurrent epidemics all over the world. Alphaviral nonstructural protein 1 (nsP1) orchestrates the capping of nascent viral RNA via its S-adenosyl methionine-dependent N-7-methyltransferase (MTase) and guanylyltransferase activities. Here, we developed and validated a novel capillary electrophoresis (CE)-based assay for measuring the MTase activity of purified VEEV and CHIKV nsP1. We employed the assay to assess the MTase inhibition efficiency of a few adenosine analogs and identified 5-iodotubercidin (5-IT) as an inhibitor of nsP1. The antiviral potency of 5-IT was evaluated in vitro using a combination of cell-based assays, which suggest that 5-IT is efficacious against CHIKV in cell culture (EC50 : 0.409 µm).
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Affiliation(s)
- Rajat Mudgal
- Department of Biotechnology, Indian Institute of Technology Roorkee, India
| | - Supreeti Mahajan
- Department of Biotechnology, Indian Institute of Technology Roorkee, India
| | - Shailly Tomar
- Department of Biotechnology, Indian Institute of Technology Roorkee, India
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Jaïs PH, Decroly E, Jacquet E, Le Boulch M, Jaïs A, Jean-Jean O, Eaton H, Ponien P, Verdier F, Canard B, Goncalves S, Chiron S, Le Gall M, Mayeux P, Shmulevitz M. C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system. Nucleic Acids Res 2019; 47:2681-2698. [PMID: 30726994 PMCID: PMC6412113 DOI: 10.1093/nar/gkz069] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2018] [Revised: 12/03/2018] [Accepted: 01/25/2019] [Indexed: 12/25/2022] Open
Abstract
Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.
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Affiliation(s)
- Philippe H Jaïs
- Eukarÿs SAS, Génopole Campus 3, 4 rue Pierre Fontaine, 91058 Evry Cedex, France
| | - Etienne Decroly
- Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257 CNRS/AMU, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France
| | - Eric Jacquet
- Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Marine Le Boulch
- Eukarÿs SAS, Génopole Campus 3, 4 rue Pierre Fontaine, 91058 Evry Cedex, France
| | - Aurélien Jaïs
- Eukarÿs SAS, Génopole Campus 3, 4 rue Pierre Fontaine, 91058 Evry Cedex, France
| | - Olivier Jean-Jean
- Sorbonne Université, CNRS-UMR8256, Biological Adaptation and Ageing, Institut de Biologie Paris Seine (B2A-IBPS), F-75252 Paris, France
| | - Heather Eaton
- Medical Microbiology and Immunology, University of Alberta, 6-142J Katz Group Centre for Pharmacy and Health Research, 114 Street NW, Edmonton, Alberta T6G 2E1, Canada
| | - Prishila Ponien
- Institut de Chimie des Substances Naturelles, CNRS UPR2301, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Fréderique Verdier
- INSERM Unit 1016, Institut Cochin, Bâtiment Gustave Roussy, 27 rue du faubourg Saint-Jacques, 75014 Paris, France
| | - Bruno Canard
- Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257 CNRS/AMU, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France
| | - Sergio Goncalves
- Eukarÿs SAS, Génopole Campus 3, 4 rue Pierre Fontaine, 91058 Evry Cedex, France
| | - Stéphane Chiron
- Eukarÿs SAS, Génopole Campus 3, 4 rue Pierre Fontaine, 91058 Evry Cedex, France
| | - Maude Le Gall
- Gastrointestinal and Metabolic Dysfunctions in Nutritional Pathologies, INSERM UMRS1149, 16 rue Henri Huchard, 75890 Paris Cedex 18, France
| | - Patrick Mayeux
- INSERM Unit 1016, Institut Cochin, Bâtiment Gustave Roussy, 27 rue du faubourg Saint-Jacques, 75014 Paris, France
| | - Maya Shmulevitz
- Medical Microbiology and Immunology, University of Alberta, 6-142J Katz Group Centre for Pharmacy and Health Research, 114 Street NW, Edmonton, Alberta T6G 2E1, Canada
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Huang YP, Huang YW, Hsiao YJ, Li SC, Hsu YH, Tsai CH. Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana. JOURNAL OF EXPERIMENTAL BOTANY 2019; 70:4657-4670. [PMID: 31552430 PMCID: PMC6760330 DOI: 10.1093/jxb/erz244] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Indexed: 05/20/2023]
Abstract
Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.
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Affiliation(s)
- Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ying-Wen Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Yung-Jen Hsiao
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Siou-Cen Li
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Yau-Huei Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Advanced Plant Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Advanced Plant Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
- Research Center for Sustainable Energy and Nanotechnology, National Chung Hsing University, Taichung, Taiwan
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11
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Chen I, Chang J, Wu C, Huang Y, Hsu Y, Tsai C. An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication. MOLECULAR PLANT PATHOLOGY 2019; 20:673-684. [PMID: 30924604 PMCID: PMC6637893 DOI: 10.1111/mpp.12784] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/19/2023]
Abstract
One up-regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. The full length cDNA of this gene was cloned by 5' and 3'-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up-regulated gene, NbUbE3R1 that plays a role in BaMV replication.
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Affiliation(s)
- I‐Hsuan Chen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Jui‐En Chang
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Chen‐Yu Wu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Ying‐Ping Huang
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Yau‐Huei Hsu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
- Advanced Plant Biotechnology CenterNational Chung Hsing UniversityTaichung402Taiwan
| | - Ching‐Hsiu Tsai
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
- Advanced Plant Biotechnology CenterNational Chung Hsing UniversityTaichung402Taiwan
- Research Center for Sustainable Energy and NanotechnologyNational Chung Hsing UniversityTaichung402Taiwan
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Lee C, Wu Y, Hsueh C, Huang Y, Hsu Y, Meng M. Mitogen-activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana. MOLECULAR PLANT PATHOLOGY 2018; 19:2319-2332. [PMID: 29806182 PMCID: PMC6638022 DOI: 10.1111/mpp.12701] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 04/22/2018] [Accepted: 05/20/2018] [Indexed: 06/08/2023]
Abstract
In plants, the mitogen-activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen-associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo mosaic virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV-encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity-dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail mosaic virus but decreased Potato virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad-spectrum resistance through genetically manipulating a single player in MAPK cascades.
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Affiliation(s)
- Cheng‐Cheng Lee
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan, ROC40227
| | - Yi‐Jhen Wu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan, ROC40227
| | - Chia‐Hsin Hsueh
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan, ROC40227
| | - Yu‐Ting Huang
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan, ROC40227
| | - Yau‐Heiu Hsu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan, ROC40227
| | - Menghsiao Meng
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan, ROC40227
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Chen I, Chen H, Huang Y, Huang H, Shenkwen L, Hsu Y, Tsai C. A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus. MOLECULAR PLANT PATHOLOGY 2018; 19:405-417. [PMID: 28052479 PMCID: PMC6637981 DOI: 10.1111/mpp.12532] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/30/2016] [Revised: 12/25/2016] [Accepted: 12/29/2016] [Indexed: 05/05/2023]
Abstract
An up-regulated gene derived from Bamboo mosaic virus (BaMV)-infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single-stranded, positive-sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active-site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus-induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post-inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2-knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2-knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2-OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co-immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.
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Affiliation(s)
- I‐Hsuan Chen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Hui‐Ting Chen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Ying‐Ping Huang
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Hui‐Chen Huang
- Biotechnology CenterNational Chung Hsing UniversityTaichung402Taiwan
| | - Lin‐Ling Shenkwen
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Yau‐Heiu Hsu
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
| | - Ching‐Hsiu Tsai
- Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichung402Taiwan
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Chen IH, Tsai AY, Huang YP, Wu IF, Cheng SF, Hsu YH, Tsai CH. Nuclear-Encoded Plastidal Carbonic Anhydrase Is Involved in Replication of Bamboo mosaic virus RNA in Nicotiana benthamiana. Front Microbiol 2017; 8:2046. [PMID: 29093706 PMCID: PMC5651272 DOI: 10.3389/fmicb.2017.02046] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Accepted: 10/06/2017] [Indexed: 01/29/2023] Open
Abstract
On inoculation of Nicotiana benthamiana with Bamboo mosaic virus (BaMV), a gene with downregulated expression was found involved in the infection cycle of BaMV. To uncover how this downregulated gene affects the accumulation of BaMV in plants, we used loss- and gain-of-function experiments. Knockdown of this gene decreased the accumulation of BaMV coat protein to approximately 60% in both plants and protoplasts of N. benthamiana but had no effect on Potato virus X and Cucumber mosaic virus infection. The full-length gene was cloned and revealed as an N. benthamiana nuclear-encoded chloroplast carbonic anhydrase (CA) and so designated NbCA. As compared with the accumulation of BaMV RNAs in NbCA-knockdown protoplasts, both plus- and minus-strand RNAs were reduced. We further fused NbCA with Orange fluorescent protein to confirm its localization in chloroplasts on confocal microscopy. However, transiently expressed NbCA in chloroplasts did not considerably increase BaMV accumulation. The addition of exogenous CA may not have any additive effect on BaMV accumulation because of the natural abundance of CA in chloroplasts. In an in vitro replication assay, the addition of Escherichia coli-expressed NbCA enhanced exogenous template level (re-initiation and elongation) but not endogenous template level (only elongation). These results suggest that NbCA is possibly involved in re-initiation step of BaMV RNA replication. Further analysis indicated that proton concentration could influence the endogenous and exogenous template activities. Hence, our results implied that NbCA could be playing a role in harnessing proton concentration and favoring the replicase with the re-initiation template.
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Affiliation(s)
- I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - April Y Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - I-Fan Wu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Shun-Fang Cheng
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.,Research Center for Sustainable Energy and Nanotechnology, National Chung Hsing University, Taichung, Taiwan
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Huang YP, Huang YW, Chen IH, Shenkwen LL, Hsu YH, Tsai CH. Plasma membrane-associated cation-binding protein 1-like protein negatively regulates intercellular movement of BaMV. JOURNAL OF EXPERIMENTAL BOTANY 2017; 68:4765-4774. [PMID: 28992255 PMCID: PMC5853580 DOI: 10.1093/jxb/erx307] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Accepted: 08/04/2017] [Indexed: 05/13/2023]
Abstract
To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement.
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Affiliation(s)
- Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Ying-Wen Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Lin-Ling Shenkwen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Yau-Huei Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
- Research Center for Sustainable Energy and Nanotechnology, National Chung Hsing University, Taichung, 402, Taiwan
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Chen IH, Huang YP, Tseng CH, Ni JT, Tsai CH, Hsu YH, Tsai CH. Nicotiana benthamiana Elicitor-Inducible Leucine-Rich Repeat Receptor-Like Protein Assists Bamboo Mosaic Virus Cell-to-Cell Movement. FRONTIERS IN PLANT SCIENCE 2017; 8:1736. [PMID: 29056941 PMCID: PMC5635722 DOI: 10.3389/fpls.2017.01736] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Accepted: 09/22/2017] [Indexed: 06/07/2023]
Abstract
For successful infection, a virus requires various host factors at different stages such as translation, targeting, replication, and spreading. One of the host genes upregulated after Nicotiana benthamiana infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA potexvirus, assists in viral movement. To understand how this host protein is involved in BaMV movement, we cloned its full-length cDNA by rapid amplification of cDNA ends. The gene has 3199 nt and encodes a 969-amino acid polypeptide. The sequence of the encoded polypeptide is orthologous to that of N. tabacum elicitor-inducible leucine-rich repeat (LRR) receptor-like protein (NtEILP), a plant viral resistance gene, and is designated NbEILP. To reveal how NbEILP is involved in BaMV movement, we fused green fluorescent protein (GFP) to its C-terminus. Unfortunately, the gene's expression in N. benthamiana was beyond our detection limit possibly because of its large size (∼135 kDa). However, NbEILP at such low expression could still enhance BaMV accumulation in inoculated leaves. A short version of NbEILP was constructed to remove the LRR domain, NbEILP/ΔLRR-GFP; the expression of this deletion mutant could still enhance BaMV accumulation to 1.7-fold that of the control. Hence, the LRR domain in NbEILP is not an essential element in BaMV movement. We constructed a few deletion mutants - NbEILP/ΔLRRΔTMD (without the transmembrane domain), NbEILP/ΔLRRΔCD (without the cytoplasmic domain), and NbEILP/ΔLRRΔSP (without the signal peptide) - to examine whether these domains are involved in BaMV movement. For BaMV movement, NbEILP requires the signal peptide to target the endoplasmic reticulum and the transmembrane domain to retain on the membrane.
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Decroly E, Canard B. Biochemical principles and inhibitors to interfere with viral capping pathways. Curr Opin Virol 2017; 24:87-96. [PMID: 28527860 PMCID: PMC7185569 DOI: 10.1016/j.coviro.2017.04.003] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2017] [Revised: 04/07/2017] [Accepted: 04/10/2017] [Indexed: 12/16/2022]
Abstract
Many viruses cap their mRNAs with their own enzymes. The latter have significantly different structures and mechanisms from cellular capping enzymes. Unique active-site architecture and mechanisms should expedite inhibitor design. Capping enzymes and/or cap-methyltransferases are designated antiviral targets. Messenger RNAs are decorated by a cap structure, which is essential for their translation into proteins. Many viruses have developed strategies in order to cap their mRNAs. The cap is either synthetized by a subset of viral or cellular enzymes, or stolen from capped cellular mRNAs by viral endonucleases (‘cap-snatching’). Reverse genetic studies provide evidence that inhibition of viral enzymes belonging to the capping pathway leads to inhibition of virus replication. The replication defect results from reduced protein synthesis as well as from detection of incompletely capped RNAs by cellular innate immunity sensors. Thus, it is now admitted that capping enzymes are validated antiviral targets, as their inhibition will support an antiviral response in addition to the attenuation of viral mRNA translation. In this review, we describe the different viral enzymes involved in mRNA capping together with relevant inhibitors, and their biochemical features useful in inhibitor discovery.
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Affiliation(s)
- Etienne Decroly
- CNRS, Aix Marseille University, AFMB UMR7257, Marseille, France.
| | - Bruno Canard
- CNRS, Aix Marseille University, AFMB UMR7257, Marseille, France.
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18
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Lin KY, Lin NS. Interfering Satellite RNAs of Bamboo mosaic virus. Front Microbiol 2017; 8:787. [PMID: 28522996 PMCID: PMC5415622 DOI: 10.3389/fmicb.2017.00787] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2017] [Accepted: 04/18/2017] [Indexed: 11/13/2022] Open
Abstract
Satellite RNAs (satRNAs) are sub-viral agents that may interact with their cognate helper virus (HV) and host plant synergistically and/or antagonistically. SatRNAs totally depend on the HV for replication, so satRNAs and HV usually evolve similar secondary or tertiary RNA structures that are recognized by a replication complex, although satRNAs and HV do not share an appreciable sequence homology. The satRNAs of Bamboo mosaic virus (satBaMV), the only satRNAs of the genus Potexvirus, have become one of the models of how satRNAs can modulate HV replication and virus-induced symptoms. In this review, we summarize the molecular mechanisms underlying the interaction of interfering satBaMV and BaMV. Like other satRNAs, satBaMV mimics the secondary structures of 5'- and 3'-untranslated regions (UTRs) of BaMV as a molecular pretender. However, a conserved apical hairpin stem loop (AHSL) in the 5'-UTR of satBaMV was found as the key determinant for downregulating BaMV replication. In particular, two unique nucleotides (C60 and C83) in the AHSL of satBaMVs determine the satBaMV interference ability by competing for the replication machinery. Thus, transgenic plants expressing interfering satBaMV could confer resistance to BaMV, and interfering satBaMV could be used as biological-control agent. Unlike two major anti-viral mechanisms, RNA silencing and salicylic acid-mediated immunity, our findings in plants by in vivo competition assay and RNA deep sequencing suggested replication competition is involved in this transgenic satBaMV-mediated BaMV interference. We propose how a single nucleotide of satBaMV can make a great change in BaMV pathogenicity and the underlying mechanism.
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Affiliation(s)
- Kuan-Yu Lin
- Institute of Plant and Microbial Biology, Academia SinicaTaipei, Taiwan
| | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia SinicaTaipei, Taiwan
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Meng M, Lee CC. Function and Structural Organization of the Replication Protein of Bamboo mosaic virus. Front Microbiol 2017; 8:522. [PMID: 28400766 PMCID: PMC5368238 DOI: 10.3389/fmicb.2017.00522] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 03/13/2017] [Indexed: 12/17/2022] Open
Abstract
The genus Potexvirus is one of the eight genera belonging to the family Alphaflexiviridae according to the Virus Taxonomy 2015 released by International Committee on Taxonomy of Viruses (www.ictvonline.org/index.asp). Currently, the genus contains 35 known species including many agricultural important viruses, e.g., Potato virus X (PVX). Members of this genus are characterized by flexuous, filamentous virions of 13 nm in diameter and 470-580 nm in length. A potexvirus has a monopartite positive-strand RNA genome, encoding five open-reading frames (ORFs), with a cap structure at the 5' end and a poly(A) tail at the 3' end. Besides PVX, Bamboo mosaic virus (BaMV) is another potexvirus that has received intensive attention due to the wealth of knowledge on the molecular biology of the virus. In this review, we discuss the enzymatic activities associated with each of the functional domains of the BaMV replication protein, a 155-kDa polypeptide encoded by ORF1. The unique cap formation mechanism, which may be conserved across the alphavirus superfamily, is particularly addressed. The recently identified interactions between the replication protein and the plant host factors are also described.
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Affiliation(s)
- Menghsiao Meng
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Cheng-Cheng Lee
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
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Huang YP, Chen IH, Tsai CH. Host Factors in the Infection Cycle of Bamboo mosaic virus. Front Microbiol 2017; 8:437. [PMID: 28360904 PMCID: PMC5350103 DOI: 10.3389/fmicb.2017.00437] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2017] [Accepted: 03/02/2017] [Indexed: 12/02/2022] Open
Abstract
To complete the infection cycle efficiently, the virus must hijack the host systems in order to benefit for all the steps and has to face all the defense mechanisms from the host. This review involves a discussion of how these positive and negative factors regulate the viral RNA accumulation identified for the Bamboo mosaic virus (BaMV), a single-stranded RNA virus. The genome of BaMV is approximately 6.4 kb in length, encoding five functional polypeptides. To reveal the host factors involved in the infection cycle of BaMV, a few different approaches were taken to screen the candidates. One of the approaches is isolating the viral replicase-associated proteins by co-immunoprecipitation with the transiently expressed tagged viral replicase in plants. Another approach is using the cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes derived from N. benthamiana plants after infection. The candidates are examined by knocking down the expression in plants using the Tobacco rattle virus-based virus-induced gene silencing technique following BaMV inoculation. The positive or negative regulators could be described as reducing or enhancing the accumulation of BaMV in plants when the expression levels of these proteins are knocked down. The possible roles of these host factors acting on the accumulation of BaMV will be discussed.
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Affiliation(s)
- Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
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21
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Parvez MK. Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity. World J Gastroenterol 2017; 23:590-602. [PMID: 28216965 PMCID: PMC5292332 DOI: 10.3748/wjg.v23.i4.590] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2016] [Revised: 12/05/2016] [Accepted: 01/04/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the role of non-structural open reading frame 1 "Y-domain" sequences in the hepatitis E virus (HEV) life cycle. METHODS Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions' infectivity was assayed on naïve HepG2/C3A cells. RESULTS In silico analysis identified a potential palmitoylation-site (C336C337) and an α-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity. CONCLUSION This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.
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Deficiency of the eIF4E isoform nCBP limits the cell-to-cell movement of a plant virus encoding triple-gene-block proteins in Arabidopsis thaliana. Sci Rep 2017; 7:39678. [PMID: 28059075 PMCID: PMC5216350 DOI: 10.1038/srep39678] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2016] [Accepted: 11/25/2016] [Indexed: 01/19/2023] Open
Abstract
One of the important antiviral genetic strategies used in crop breeding is recessive resistance. Two eukaryotic translation initiation factor 4E family genes, eIF4E and eIFiso4E, are the most common recessive resistance genes whose absence inhibits infection by plant viruses in Potyviridae, Carmovirus, and Cucumovirus. Here, we show that another eIF4E family gene, nCBP, acts as a novel recessive resistance gene in Arabidopsis thaliana toward plant viruses in Alpha- and Betaflexiviridae. We found that infection by Plantago asiatica mosaic virus (PlAMV), a potexvirus, was delayed in ncbp mutants of A. thaliana. Virus replication efficiency did not differ between an ncbp mutant and a wild type plant in single cells, but viral cell-to-cell movement was significantly delayed in the ncbp mutant. Furthermore, the accumulation of triple-gene-block protein 2 (TGB2) and TGB3, the movement proteins of potexviruses, decreased in the ncbp mutant. Inoculation experiments with several viruses showed that the accumulation of viruses encoding TGBs in their genomes decreased in the ncbp mutant. These results indicate that nCBP is a novel member of the eIF4E family recessive resistance genes whose loss impairs viral cell-to-cell movement by inhibiting the efficient accumulation of TGB2 and TGB3.
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Lin W, Wang L, Yan W, Chen L, Chen H, Yang W, Guo M, Wu Z, Yang L, Xie L. Identification and characterization of Bamboo mosaic virus isolates from a naturally occurring coinfection in Bambusa xiashanensis. Arch Virol 2017; 162:1335-1339. [PMID: 28050737 DOI: 10.1007/s00705-016-3191-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Accepted: 11/20/2016] [Indexed: 12/26/2022]
Abstract
Bamboo mosaic virus (BaMV) is a well-characterized virus and a model of virus-host interaction in plants. Here, we identified naturally occurring BaMV isolates from Fujian Province, China and furthermore describe a naturally occurring BaMV coinfection in bamboo (Bambusa xiashanensis) plants. Two different types of BaMV were identified, represented by isolates BaMV-XSNZHA7 (X7) and BaMV-XSNZHA10 (X10). The phylogenetic relationships between X7- and X10-like isolates and published BaMV isolates were determined based on genomic RNA and amino acid sequences. Three clusters were identified, indicating that BaMV is highly diverse. The in planta viral replication kinetics were determined for X7 and X10 in single infections and in an X7/X10 coinfection. The peak viral load during coinfection was significantly greater than that during single infection with either virus and contained a slightly higher proportion of X10 virus than X7, suggesting that X10-like viruses may have a fitness advantage when compared to X7-like viruses.
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Affiliation(s)
- Wenwu Lin
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Lu Wang
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenkai Yan
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Lingli Chen
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Huihuang Chen
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenting Yang
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Maohui Guo
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Zujian Wu
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
| | - Liang Yang
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
| | - Lianhui Xie
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
- Fujian Key Laboratory of Plant Virology, Virology, Institute of Plant Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
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Alazem M, Lin NS. Antiviral Roles of Abscisic Acid in Plants. FRONTIERS IN PLANT SCIENCE 2017; 8:1760. [PMID: 29075279 PMCID: PMC5641568 DOI: 10.3389/fpls.2017.01760] [Citation(s) in RCA: 79] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Accepted: 09/26/2017] [Indexed: 05/18/2023]
Abstract
Abscisic acid (ABA) is a key hormone involved in tuning responses to several abiotic stresses and also has remarkable impacts on plant defense against various pathogens. The roles of ABA in plant defense against bacteria and fungi are multifaceted, inducing or reducing defense responses depending on its time of action. However, ABA induces different resistance mechanisms to viruses regardless of the induction time. Recent studies have linked ABA to the antiviral silencing pathway, which interferes with virus accumulation, and the micro RNA (miRNA) pathway through which ABA affects the maturation and stability of miRNAs. ABA also induces callose deposition at plasmodesmata, a mechanism that limits viral cell-to-cell movement. Bamboo mosaic virus (BaMV) is a member of the potexvirus group and is one of the most studied viruses in terms of the effects of ABA on its accumulation and resistance. In this review, we summarize how ABA interferes with the accumulation and movement of BaMV and other viruses. We also highlight aspects of ABA that may have an effect on other types of resistance and that require further investigation.
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Lin W, Yan W, Yang W, Yu C, Chen H, Zhang W, Wu Z, Yang L, Xie L. Characterisation of siRNAs derived from new isolates of bamboo mosaic virus and their associated satellites in infected ma bamboo (Dendrocalamus latiflorus). Arch Virol 2016; 162:505-510. [PMID: 27743256 DOI: 10.1007/s00705-016-3092-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2016] [Accepted: 09/26/2016] [Indexed: 12/24/2022]
Abstract
We characterised the virus-derived small interfering RNAs (vsiRNA) of bamboo mosaic virus (Ba-vsiRNAs) and its associated satellite RNA (satRNA)-derived siRNAs (satsiRNAs) in a bamboo plant (Dendrocalamus latiflorus) by deep sequencing. Ba-vsiRNAs and satsiRNAs of 21-22 nt in length, with both (+) and (-) polarity, predominated. The 5'-terminal base of Ba-vsiRNA was biased towards A, whereas a bias towards C/U was observed in sense satsiRNAs, and towards A in antisense satsiRNAs. A large set of bamboo genes were identified as potential targets of Ba-vsiRNAs and satsiRNAs, revealing RNA silencing-based virus-host interactions in plants. Moreover, we isolated and characterised new isolates of bamboo mosaic virus (BaMV; 6,350 nt) and BaMV-associated satRNA (satBaMV; 834 nt), designated BaMV-MAZSL1 and satBaMV-MAZSL1, respectively.
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Affiliation(s)
- Wenwu Lin
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenkai Yan
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenting Yang
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Chaowei Yu
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Huihuang Chen
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wen Zhang
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Zujian Wu
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Liang Yang
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
| | - Lianhui Xie
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
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Huang YP, Jhuo JH, Tsai MS, Tsai CH, Chen HC, Lin NS, Hsu YH, Cheng CP. NbRABG3f, a member of Rab GTPase, is involved in Bamboo mosaic virus infection in Nicotiana benthamiana. MOLECULAR PLANT PATHOLOGY 2016; 17:714-26. [PMID: 26416342 PMCID: PMC6638505 DOI: 10.1111/mpp.12325] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up-regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up-regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus-induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane-targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP-NbRABG3f/T22N clearly co-localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane-targeting ability are crucial for BaMV accumulation at the cell level.
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Affiliation(s)
- Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Jia-Hua Jhuo
- Department of Life Sciences, Tzu Chi University, Hualien, 970, Taiwan
| | - Meng-Shan Tsai
- Department of Life Sciences, Tzu Chi University, Hualien, 970, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Hong-Chi Chen
- Department of Life Sciences, Tzu Chi University, Hualien, 970, Taiwan
| | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Nankang Taipei, 115, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 402, Taiwan
| | - Chi-Ping Cheng
- Department of Life Sciences, Tzu Chi University, Hualien, 970, Taiwan
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Lin W, Gao F, Yang W, Yu C, Zhang J, Chen L, Wu Z, Hsu YH, Xie L. Molecular characterization and detection of a recombinant isolate of bamboo mosaic virus from China. Arch Virol 2016; 161:1091-4. [PMID: 26923929 DOI: 10.1007/s00705-016-2773-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2015] [Accepted: 01/25/2016] [Indexed: 11/30/2022]
Abstract
The complete genome sequences of three isolates of bamboo mosaic virus (BaMV) from mainland China were determined and compared to those of BaMV isolates from Taiwan. Sequence analysis showed that isolate BaMV-JXYBZ1 from Fuzhou shares 98 % nucleotide sequence identity with BaMV-YTHSL14 from nucleotides 2586 to 6306, and more than 94 % nucleotide sequence identity with BaMV-MUZHUBZ2 in other regions. Recombination and phylogenetic analyses indicate that BaMV-JXYBZ1 is a recombinant with one recombination breakpoint. To our knowledge, this is the first report of a BaMV recombinant worldwide.
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Affiliation(s)
- Wenwu Lin
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Fangluan Gao
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Wenting Yang
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Chaowei Yu
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Jie Zhang
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Lingli Chen
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Zujian Wu
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan.
| | - Lianhui Xie
- Fujian Key Lab of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian.
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Lee CC, Lin TL, Lin JW, Han YT, Huang YT, Hsu YH, Meng M. Promotion of Bamboo Mosaic Virus Accumulation in Nicotiana benthamiana by 5'→3' Exonuclease NbXRN4. Front Microbiol 2016; 6:1508. [PMID: 26779163 PMCID: PMC4702010 DOI: 10.3389/fmicb.2015.01508] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Accepted: 12/14/2015] [Indexed: 12/11/2022] Open
Abstract
Bamboo mosaic virus (BaMV) has a 6.4-kb (+) sense RNA genome with a 5' cap and a 3' poly(A) tail. ORF1 of this potexvirus encodes a 155-kDa replication protein responsible for the viral RNA replication/transcription and 5' cap formation. To learn more about the replication complex of BaMV, a protein preparation enriched in the 155-kDa replication protein was obtained from Nicotiana benthamiana by a protocol involving agroinfiltration and immunoprecipitation. Subsequent analysis by SDS-PAGE and mass spectrometry identified a handful of host proteins that may participate in the viral replication. Among them, the cytoplasmic exoribonuclease NbXRN4 particularly caught our attention. NbXRN4 has been shown to have an antiviral activity against Tomato bushy stunt virus and Tomato mosaic virus. In Arabidopsis, the enzyme could reduce RNAi- and miRNA-mediated RNA decay. This study found that downregulation of NbXRN4 greatly decreased BaMV accumulation, while overexpression of NbXRN4 resulted in an opposite effect. Mutations at the catalytically essential residues abolished the function of NbXRN4 in the increase of BaMV accumulation. Nonetheless, NbXRN4 was still able to promote BaMV accumulation in the presence of the RNA silencing suppressor P19. In summary, the replication efficiency of BaMV may be improved by the exoribonuclease activity of NbXRN4.
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Affiliation(s)
- Cheng-Cheng Lee
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Tzu-Ling Lin
- Graduate Institute of Biotechnology, National Chung Hsing UniversityTaichung, Taiwan; Division of Medicine Centre for Nephrology, University College LondonLondon, UK
| | - Jhe-Wei Lin
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Yu-Tsung Han
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Yu-Ting Huang
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
| | - Menghsiao Meng
- Graduate Institute of Biotechnology, National Chung Hsing University Taichung, Taiwan
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Ahola T, Karlin DG. Sequence analysis reveals a conserved extension in the capping enzyme of the alphavirus supergroup, and a homologous domain in nodaviruses. Biol Direct 2015; 10:16. [PMID: 25886938 PMCID: PMC4392871 DOI: 10.1186/s13062-015-0050-0] [Citation(s) in RCA: 76] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2014] [Accepted: 03/24/2015] [Indexed: 12/16/2022] Open
Abstract
Background Members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis E virus and rubella virus. They encode a capping enzyme with methyltransferase-guanylyltransferase (MTase-GTase) activity, which is an attractive drug target owing to its unique mechanism. However, its experimental study has proven very difficult. Results We examined over 50 genera of viruses by sequence analyses. Earlier studies showed that the MTase-GTase contains a “Core” region conserved in sequence. We show that it is followed by a long extension, which we termed “Iceberg” region, whose secondary structure, but not sequence, is strikingly conserved throughout the alphavirus supergroup. Sequence analyses strongly suggest that the minimal capping domain corresponds to the Core and Iceberg regions combined, which is supported by earlier experimental data. The Iceberg region contains all known membrane association sites that contribute to the assembly of viral replication factories. We predict that it may also contain an overlooked, widely conserved membrane-binding amphipathic helix. Unexpectedly, we detected a sequence homolog of the alphavirus MTase-GTase in taxa related to nodaviruses and to chronic bee paralysis virus. The presence of a capping enzyme in nodaviruses is biologically consistent, since they have capped genomes but replicate in the cytoplasm, where no cellular capping enzyme is present. The putative MTase-GTase domain of nodaviruses also contains membrane-binding sites that may drive the assembly of viral replication factories, revealing an unsuspected parallel with the alphavirus supergroup. Conclusions Our work will guide the functional analysis of the alphaviral MTase-GTase and the production of domains for structure determination. The identification of a homologous domain in a simple model system, nodaviruses, which replicate in numerous eukaryotic cell systems (yeast, flies, worms, mammals, and plants), can further help crack the function and structure of the enzyme. Reviewers This article was reviewed by Valerian Dolja, Eugene Koonin and Sebastian Maurer-Stroh. Electronic supplementary material The online version of this article (doi:10.1186/s13062-015-0050-0) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Tero Ahola
- Department of Food and Environmental Sciences, University of Helsinki, 00014, Helsinki, Finland.
| | - David G Karlin
- Department of Zoology, University of Oxford, Oxford, OX1 3PS, UK. .,The Division of Structural Biology, Henry Wellcome Building, Roosevelt Drive, Oxford, OX3 7BN, UK.
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Hung CJ, Hu CC, Lin NS, Lee YC, Meng M, Tsai CH, Hsu YH. Two key arginine residues in the coat protein of Bamboo mosaic virus differentially affect the accumulation of viral genomic and subgenomic RNAs. MOLECULAR PLANT PATHOLOGY 2014; 15:196-210. [PMID: 24393453 PMCID: PMC6638855 DOI: 10.1111/mpp.12080] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The interactions between viral RNAs and coat proteins (CPs) are critical for the efficient completion of infection cycles of RNA viruses. However, the specificity of the interactions between CPs and genomic or subgenomic RNAs remains poorly understood. In this study, Bamboo mosaic virus (BaMV) was used to analyse such interactions. Using reversible formaldehyde cross-linking and mass spectrometry, two regions in CP, each containing a basic amino acid (R99 and R227, respectively), were identified to bind directly to the 5' untranslated region of BaMV genomic RNA. Analyses of the alanine mutations of R99 and R227 revealed that the secondary structures of CP were not affected significantly, whereas the accumulation of BaMV genomic, but not subgenomic, RNA was severely decreased at 24 h post-inoculation in the inoculated protoplasts. In the absence of CP, the accumulation levels of genomic and subgenomic RNAs were decreased to 1.1%-1.5% and 33%-40% of that of the wild-type (wt), respectively, in inoculated leaves at 5 days post-inoculation (dpi). In contrast, in the presence of mutant CPs, the genomic RNAs remained about 1% of that of wt, whereas the subgenomic RNAs accumulated to at least 87%, suggesting that CP might increase the accumulation of subgenomic RNAs. The mutations also restricted viral movement and virion formation in Nicotiana benthamiana leaves at 5 dpi. These results demonstrate that R99 and R227 of CP play crucial roles in the accumulation, movement and virion formation of BaMV RNAs, and indicate that genomic and subgenomic RNAs interact differently with BaMV CP.
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Affiliation(s)
- Chien-Jen Hung
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
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Cheng SF, Huang YP, Chen LH, Hsu YH, Tsai CH. Chloroplast phosphoglycerate kinase is involved in the targeting of Bamboo mosaic virus to chloroplasts in Nicotiana benthamiana plants. PLANT PHYSIOLOGY 2013; 163:1598-608. [PMID: 24154620 PMCID: PMC3846135 DOI: 10.1104/pp.113.229666] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/02/2013] [Accepted: 10/22/2013] [Indexed: 05/18/2023]
Abstract
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.
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Affiliation(s)
| | | | - Li-Hung Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan (S.-F.C., Y.-P.H., L.-H.C., Y.-H.H., C.-H.T.); and
- Graduate Institute of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan (Y.-H.H., C.-H.T.)
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan (S.-F.C., Y.-P.H., L.-H.C., Y.-H.H., C.-H.T.); and
- Graduate Institute of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan (Y.-H.H., C.-H.T.)
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Park MR, Seo JK, Kim KH. Viral and nonviral elements in potexvirus replication and movement and in antiviral responses. Adv Virus Res 2013; 87:75-112. [PMID: 23809921 DOI: 10.1016/b978-0-12-407698-3.00003-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
In Potato virus X, a member of the genus Potexvirus, special sequences and structures at the 5' and 3' ends of the nontranslated region function as cis-acting elements for viral replication. These elements greatly affect interactions between viral RNAs and those between viral RNAs and host factors. The potexvirus genome encodes five open-reading frames. Viral replicase, which is required for the synthesis of viral RNA, binds viral RNA elements and host factors to form a viral replication complex at the host cellular membrane. The coat protein (CP) and three viral movement proteins (TGB1, TGB2, and TGB3) have critical roles in mediating cell-to-cell viral movement through plasmodesmata by virion formation or by nonvirion ribonucleoprotein (RNP) complex formation with viral movement proteins (TGBs). The RNP complex, like TGB1-CP-viral RNA, is associated with viral replicase and used for immediate reinitiation of viral replication in newly invaded cells. Higher plants have defense mechanisms against potexviruses such as Rx-mediated resistance and RNA silencing. The CP acts as an avirulence effector for plant defense mechanisms, while TGB1 functions as a viral suppressor of RNA silencing, which is the mechanism of innate immune resistance. Here, we describe recent findings concerning the involvement of viral and host factors in potexvirus replication and in antiviral responses to potexvirus infection.
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Affiliation(s)
- Mi-Ri Park
- Department of Agricultural Biotechnology, Plant Genomics and Breeding Institute, Seoul National University, Seoul, Republic of Korea
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Chen IH, Chiu MH, Cheng SF, Hsu YH, Tsai CH. The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus. THE NEW PHYTOLOGIST 2013; 199:749-57. [PMID: 23701112 PMCID: PMC3744755 DOI: 10.1111/nph.12304] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Accepted: 03/28/2013] [Indexed: 05/23/2023]
Abstract
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.
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Affiliation(s)
- I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing UniversityTaichung, 402, Taiwan
| | - Meng-Hsuen Chiu
- Graduate Institute of Biotechnology, National Chung Hsing UniversityTaichung, 402, Taiwan
| | - Shun-Fang Cheng
- Graduate Institute of Biotechnology, National Chung Hsing UniversityTaichung, 402, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing UniversityTaichung, 402, Taiwan
- Graduate Institute of Medical Laboratory Science and Biotechnology, China Medical UniversityTaichung, 404, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing UniversityTaichung, 402, Taiwan
- Graduate Institute of Medical Laboratory Science and Biotechnology, China Medical UniversityTaichung, 404, Taiwan
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Chou YL, Hung YJ, Tseng YH, Hsu HT, Yang JY, Wung CH, Lin NS, Meng M, Hsu YH, Chang BY. The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus. PLoS Pathog 2013; 9:e1003405. [PMID: 23754943 PMCID: PMC3675025 DOI: 10.1371/journal.ppat.1003405] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2012] [Accepted: 04/22/2013] [Indexed: 12/03/2022] Open
Abstract
The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.
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Affiliation(s)
- Yuan-Lin Chou
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Yi-Jing Hung
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Yang-Hao Tseng
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Hsiu-Ting Hsu
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Jun-Yi Yang
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Chiung-Hua Wung
- Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China
| | - Menghsiao Meng
- Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China
| | - Ban-Yang Chang
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China
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Cheng SF, Tsai MS, Huang CL, Huang YP, Chen IH, Lin NS, Hsu YH, Tsai CH, Cheng CP. Ser/Thr kinase-like protein of Nicotiana benthamiana is involved in the cell-to-cell movement of Bamboo mosaic virus. PLoS One 2013; 8:e62907. [PMID: 23646157 PMCID: PMC3639906 DOI: 10.1371/journal.pone.0062907] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2012] [Accepted: 03/28/2013] [Indexed: 01/08/2023] Open
Abstract
To investigate the plant genes affected by Bamboo mosaic virus (BaMV) infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL) gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV) and Potato virus X (PVX); thus, NbSTKL might be a specific protein facilitating BaMV movement.
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Affiliation(s)
- Shun-Fang Cheng
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Meng-Shan Tsai
- Department of Life Sciences, Tzu Chi University, Hualien, Taiwan
| | - Chia-Lin Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Nankang Taipei, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Chi-Ping Cheng
- Department of Life Sciences, Tzu Chi University, Hualien, Taiwan
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Ferron F, Decroly E, Selisko B, Canard B. The viral RNA capping machinery as a target for antiviral drugs. Antiviral Res 2012; 96:21-31. [PMID: 22841701 PMCID: PMC7114304 DOI: 10.1016/j.antiviral.2012.07.007] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2012] [Revised: 07/09/2012] [Accepted: 07/13/2012] [Indexed: 12/18/2022]
Abstract
Most viruses modify their genomic and mRNA 5′-ends with the addition of an RNA cap, allowing efficient mRNA translation, limiting degradation by cellular 5′–3′ exonucleases, and avoiding its recognition as foreign RNA by the host cell. Viral RNA caps can be synthesized or acquired through the use of a capping machinery which exhibits a significant diversity in organization, structure and mechanism relative to that of their cellular host. Therefore, viral RNA capping has emerged as an interesting field for antiviral drug design. Here, we review the different pathways and mechanisms used to produce viral mRNA 5′-caps, and present current structures, mechanisms, and inhibitors known to act on viral RNA capping.
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Affiliation(s)
- François Ferron
- Centre National de la Recherche Scientifique and Aix-Marseille Université, UMR 7257, Architecture et Fonction des Macromolécules Biologiques, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France
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Lin HY, Yu CY, Hsu YH, Meng M. Functional analysis of the conserved histidine residue of Bamboo mosaic virus capping enzyme in the activity for the formation of the covalent enzyme-m7GMP intermediate. FEBS Lett 2012; 586:2326-31. [PMID: 22641040 DOI: 10.1016/j.febslet.2012.05.024] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2012] [Revised: 05/11/2012] [Accepted: 05/11/2012] [Indexed: 10/28/2022]
Abstract
The alphavirus-like mRNA capping enzyme of Bamboo mosaic virus (BaMV) exhibits an AdoMet-dependent guanylyltransferase activity by which the methyl group of AdoMet is transferred to GTP, leading to the formation of m(7)GTP, and the m(7)GMP moiety is next transferred to the 5' end of ppRNA via a covalent enzyme-m(7)GMP intermediate. The function of the conserved H68 of the BaMV capping enzyme in the intermediate formation was analyzed by mutagenesis in this study. The nature of the bond linking the enzyme and m(7)GMP was changed in the H68C mutant protein, strongly suggesting that H68 covalently binds to m(7)GMP in the intermediate.
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Affiliation(s)
- Hua-Yang Lin
- Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 40227, Taiwan, ROC
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Huang YW, Hu CC, Liou MR, Chang BY, Tsai CH, Meng M, Lin NS, Hsu YH. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA. PLoS Pathog 2012; 8:e1002726. [PMID: 22654666 PMCID: PMC3359997 DOI: 10.1371/journal.ppat.1002726] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2011] [Accepted: 04/16/2012] [Indexed: 12/22/2022] Open
Abstract
Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR) of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.
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Affiliation(s)
- Ying Wen Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
| | - Chung Chi Hu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ming Ru Liou
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ban Yang Chang
- Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan
| | - Ching Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Menghsiao Meng
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Na Sheng Lin
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
| | - Yau Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
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Chen HC, Kong LR, Yeh TY, Cheng CP, Hsu YH, Lin NS. The conserved 5' apical hairpin stem loops of bamboo mosaic virus and its satellite RNA contribute to replication competence. Nucleic Acids Res 2012; 40:4641-52. [PMID: 22278884 PMCID: PMC3378871 DOI: 10.1093/nar/gks030] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2011] [Revised: 12/30/2011] [Accepted: 01/05/2012] [Indexed: 11/29/2022] Open
Abstract
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 (C(60) and C(83)) 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 (C(64) and C(86)) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C(86) was essential for BaMV replication, and that replacement of C(64) 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.
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Affiliation(s)
- Hsin-Chuan Chen
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei and Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Lih-Ren Kong
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei and Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Ting-Yu Yeh
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei and Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Chi-Ping Cheng
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei and Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Yau-Heiu Hsu
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei and Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
| | - Na-Sheng Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei and Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
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Decroly E, Ferron F, Lescar J, Canard B. Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 2011; 10:51-65. [PMID: 22138959 PMCID: PMC7097100 DOI: 10.1038/nrmicro2675] [Citation(s) in RCA: 364] [Impact Index Per Article: 26.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
mRNAs are protected at their 5′ ends by a cap structure consisting of an N7-methylated GTP molecule linked to the first transcribed nucleotide by a 5′–5′ triphosphate bond. The cap structure is essential for RNA splicing, export and stability, and allows the ribosomal complex to recognize mRNAs and ensure their efficient translation. Uncapped RNA molecules are degraded in cytoplasmic granular compartments called processing bodies and may be detected as 'non-self' by the host cell, triggering antiviral innate immune responses through the production of interferons. Conventional RNA capping (that is, of mRNAs from the host cell and from DNA viruses) requires hydrolysis of the 5′ γ-phosphate of RNA by an RNA triphosphatase, transfer of a GMP molecule onto the 5′-end of RNA by a guanylyltransferase, and methylation of this guanosine by an (guanine-N7)-methyltransferase. Subsequent methylations on the first and second transcribed nucleotides by (nucleoside-2′-O)-methyltransferases form cap-1 and cap-2 structures. Viruses have evolved highly diverse capping mechanisms to acquire cap structures using their own or cellular capping machineries, or by stealing cap structures from cellular mRNAs. Virally encoded RNA-capping machineries are diverse in terms of their genetic components, protein domain organization, enzyme structures, and reaction mechanisms and pathways, making viral RNA capping an attractive target for antiviral-drug design. Capping the 5′ end of eukaryotic mRNAs with a 7-methylguanosine moiety enables efficient splicing, nuclear export and translation of mRNAs, and also limits their degradation by cellular exonucleases. Here, Canard and colleagues describe how viruses synthesize their own mRNA cap structures or steal them from host mRNAs, allowing efficient synthesis of viral proteins and avoidance of host innate immune responses. In the eukaryotic cell, capping of mRNA 5′ ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5′–3′ exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5′ ends with either a covalently attached peptide or a cap moiety (7-methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5′-caps and escape innate immunity.
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Affiliation(s)
- Etienne Decroly
- Centre National de Recherche Scientifique and Aix-Marseille Université, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, 163 avenue de Luminy, 13288 Marseille cedex 09, France
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The interaction between bamboo mosaic virus replication protein and coat protein is critical for virus movement in plant hosts. J Virol 2011; 85:12022-31. [PMID: 21917973 DOI: 10.1128/jvi.05595-11] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
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.
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Hu RH, Lin MC, Hsu YH, Meng M. Mutational effects of the consensus aromatic residues in the mRNA capping domain of Bamboo mosaic virus on GTP methylation and virus accumulation. Virology 2011; 411:15-24. [PMID: 21227477 DOI: 10.1016/j.virol.2010.12.022] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2010] [Revised: 10/29/2010] [Accepted: 12/14/2010] [Indexed: 10/18/2022]
Abstract
RNA viruses classified in the alphavirus-like superfamily possess a distinct capping domain, catalyzing GTP methylation and subsequent transfer of the m(7)GMP moiety from m(7)GTP to the 5'-diphosphate end of viral RNA. The H68A mutation in the capping domain of Bamboo mosaic virus enhanced GTP methylation but disabled the following transguanylation, making it possible to characterize the enzyme's methyltransferase activity separately. To explore the involvement of aromatic amino acids in substrate recognition, consensus aromatic residues in the viral domain were subjected to mutational analysis in the background of H68A. Several residues, including Y126, F144, F161, Y192, Y203, Y213, and W222, were found to be critical for GTP methylation and S-adenosylmethionine (AdoMet) binding. These mutations, except for Y213, also adversely affected the GTP binding, but less extensively. In general, the mutations decreasing the activity for GTP methylation also had correspondingly detrimental effects on virus accumulation.
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Affiliation(s)
- Rei-Hsing Hu
- Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, Taiwan 40227, ROC
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Cheng SF, Huang YP, Wu ZR, Hu CC, Hsu YH, Tsai CH. Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism. BMC PLANT BIOLOGY 2010; 10:286. [PMID: 21184690 PMCID: PMC3024324 DOI: 10.1186/1471-2229-10-286] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/25/2009] [Accepted: 12/27/2010] [Indexed: 05/03/2023]
Abstract
BACKGROUND 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). RESULTS 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. CONCLUSIONS 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.
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Affiliation(s)
- Shun-Fang Cheng
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Ying-Ping Huang
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Zi-Rong Wu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Chung-Chi Hu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
| | - Yau-Heiu Hsu
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
- Graduate Institute of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, 404, Taiwan
| | - Ching-Hsiu Tsai
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan
- Graduate Institute of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, 404, Taiwan
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44
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Lan P, Yeh WB, Tsai CW, Lin NS. A unique glycine-rich motif at the N-terminal region of Bamboo mosaic virus coat protein is required for symptom expression. MOLECULAR PLANT-MICROBE INTERACTIONS : MPMI 2010; 23:903-14. [PMID: 20521953 DOI: 10.1094/mpmi-23-7-0903] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2023]
Abstract
The coat proteins (CP) of many plant viruses are multifunctional proteins. We used N-terminal sequencing and mass spectrometry/mass spectrometry analysis to identify a truncated form of the Bamboo mosaic virus (BaMV) CP missing the N-terminal 35 amino acids (N35). The N35 region is unique in the potexviruses by its containing a glycine-rich motif (GRM) not present in databases but highly conserved among BaMV isolates. Results from site-directed mutagenesis and deletion mutational analysis showed that loss of this region converted necrotic local lesions to chlorotic local lesions on Chenopodium quinoa leaves. Furthermore, this region is required for successful development of mosaic symptoms on Nicotiana benthamiana leaves but is dispensable for BaMV replication and cell-to-cell and long-distance movement as well as virion assembly. This unique GRM-containing region of BaMV CP may be a symptom determinant in specific hosts.
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Affiliation(s)
- Ping Lan
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China
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45
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RNA 5'-triphosphatase activity of the hepatitis E virus helicase domain. J Virol 2010; 84:9637-41. [PMID: 20592074 DOI: 10.1128/jvi.00492-10] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Hepatitis E virus (HEV) has a positive-sense RNA genome with a 5'-m7G cap. HEV open reading frame 1 (ORF1) encodes a polyprotein with multiple enzyme domains required for replication. HEV helicase is a nucleoside triphosphatase (NTPase) with the ability to unwind RNA duplexes in the 5'-to-3' direction. When incubated with 5'-[gamma-(32)P]RNA and 5'-[alpha-(32)P]RNA, HEV helicase released (32)P only from 5'-[gamma-(32)P]RNA, showing specificity for the gamma-beta-triphosphate bond. Removal of gamma-phosphate from the 5' end of the primary transcripts (pppRNA to ppRNA) by RNA triphosphatase is an essential step during cap formation. It is suggested that HEV employs the helicase to mediate the first step of 5' cap synthesis.
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46
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Chen IH, Lin JW, Chen YJ, Wang ZC, Liang LF, Meng M, Hsu YH, Tsai CH. The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis. MOLECULAR PLANT PATHOLOGY 2010; 11:203-12. [PMID: 20447270 PMCID: PMC6640325 DOI: 10.1111/j.1364-3703.2009.00597.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.
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Affiliation(s)
- I-Hsuan Chen
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan
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Draghici HK, Varrelmann M. Evidence that the linker between the methyltransferase and helicase domains of potato virus X replicase is involved in homologous RNA recombination. J Virol 2009; 83:7761-9. [PMID: 19439477 PMCID: PMC2708637 DOI: 10.1128/jvi.00179-08] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2008] [Accepted: 05/07/2009] [Indexed: 12/29/2022] Open
Abstract
Recombination in RNA viruses, one of the main factors contributing to their genetic variability and evolution, is a widespread phenomenon. In this study, an in vivo assay to characterize RNA recombination in potato virus X (PVX), under high selection pressure, was established. Agrobacterium tumefaciens was used to express in Nicotiana benthamiana leaf tissue both a PVX isolate labeled with green fluorescent protein (GFP) containing a coat protein deletion mutation (DeltaCP) and a transcript encoding a functional coat protein +3'-ntr. Coexpression of the constructs led to virus movement and systemic infection; reconstituted recombinants were observed in 92% of inoculated plants. Similar results were obtained using particle bombardment, demonstrating that recombination mediated by A. tumefaciens was not responsible for the occurrence of PXC recombinants. The speed of recombination could be estimated by agroinfection of two PVX mutants lacking the 3' and 5' halves of the genome, respectively, with an overlap in the triple gene block 1 gene, allowing GFP expression only in the case of recombination. Ten different pentapeptide insertion scanning replicase mutants with replication abilities comparable to wild-type virus were applied in the different recombination assays. Two neighboring mutants affecting the linker between the methyltransferase and helicase domains were shown to be strongly debilitated in their ability to recombine. The possible functional separation of replication and recombination in the replicase molecule supports the model that RNA recombination represents a distinct function of this protein, although the underlying mechanism still needs to be investigated.
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Affiliation(s)
- Heidrun-Katharina Draghici
- Department of Crop Sciences, Section Plant Virology, University of Göttingen, Grisebachstrasse 6, D-37077 Göttingen, Germany
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Cheng CW, Hsiao YY, Wu HC, Chuang CM, Chen JS, Tsai CH, Hsu YH, Wu YC, Lee CC, Meng M. Suppression of bamboo mosaic virus accumulation by a putative methyltransferase in Nicotiana benthamiana. J Virol 2009; 83:5796-805. [PMID: 19297487 PMCID: PMC2681968 DOI: 10.1128/jvi.02471-08] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2008] [Accepted: 03/10/2009] [Indexed: 12/12/2022] Open
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.
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Affiliation(s)
- Chun-Wei Cheng
- Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
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Draghici HK, Pilot R, Thiel H, Varrelmann M. Functional mapping of PVX RNA-dependent RNA-replicase using pentapeptide scanning mutagenesis-Identification of regions essential for replication and subgenomic RNA amplification. Virus Res 2009; 143:114-24. [PMID: 19463728 DOI: 10.1016/j.virusres.2009.03.013] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2008] [Revised: 03/23/2009] [Accepted: 03/25/2009] [Indexed: 12/31/2022]
Abstract
The replicase protein of Potato virus X (PVX), type species of the genus Potexvirus, was selected to identify regions essential for replication and subgenomic RNA synthesis. Replicase amino acid (aa) sequence alignment of 16 Potexvirus species resulted in the detection of overall sequence homology of 34.4-65.4%. Two regions of consensus with a high proportion of conserved aa (1-411 and 617-1437 according to PVX) were separated by a hyper-variable linker region. Pentapeptide scanning (PS) mutagenesis in a PVX full-length clone expressing green fluorescent protein (GFP) was carried out. For 69 selected PS-mutants where insertions were spread randomly over the replicase ORF the position of the insertion was determined. The replication activity was evaluated by GFP expression from subgenomic viral RNA of PVX replicase mutants. Only one functional PS-mutant was detected in the N-terminal 430 aa, containing the conserved methyltransferase domain of the protein. In the linker region from aa 430-595, nine mutations were discovered which did not induce significant effects on the replicase replication ability. The part of the protein including helicase and polymerase domains was highly intolerant for the PS insertion as demonstrated by 24 independent more or less uniformly spread mutants.
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Affiliation(s)
- Heidrun-Katharina Draghici
- Department of Crop Sciences, Section Plant Virology, University of Göttingen, Grisebachstrasse 6, D-37077 Göttingen, Germany
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Chen HC, Hsu YH, Lin NS. Downregulation of Bamboo mosaic virus replication requires the 5' apical hairpin stem loop structure and sequence of satellite RNA. Virology 2007; 365:271-84. [PMID: 17482233 DOI: 10.1016/j.virol.2007.03.050] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2006] [Revised: 12/13/2006] [Accepted: 03/28/2007] [Indexed: 10/23/2022]
Abstract
Satellite RNAs associated with Bamboo mosaic virus (satBaMV) exhibit different phenotypes. Some isolates could reduce the accumulation of BaMV RNA and attenuate the BaMV-induced symptoms in co-inoculated plants. The determinants of the downregulation of BaMV replication were mapped in the 5' hypervariable region of satBaMV, which folds into a conserved apical hairpin stem loop (AHSL) structure comprising an apical loop and two internal loops, as evidenced by enzymatic probing. We also demonstrated that the integrity of the AHSL structure of interfering satBaMV was essential for the interference of BaMV accumulation. Concurrent analyses of natural satBaMV isolates revealed that all of the interfering isolates contained the same structures and sequences in the internal loops. Further, refined analyses indicated that, besides the AHSL structure, specific nucleotides in the internal loops play a crucial role in the downregulation, which implies that they may be required for the interaction of viral/cellular factors in this process.
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Affiliation(s)
- Hsin-Chuan Chen
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC
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