Synthesis of Figwort Mosaic Virus (FMV) Coat Protein-Reverse Transcriptase Fusion Protein in Reticulocyte Lysates from a Full-length Transcript

Lisianthus (Eustoma exaltatum (L.) Salisb. ex G. Don subsp. russellianum (Hook.) Kartesz) is an economically important ornamental crop in Taiwan. Over the past decade, nine viruses have been identified or detected in lisianthus including: Bean yellow mosaic virus (BYMV), Lisianthus necrosis virus (LNV) (2), Cucumber mosaic virus (CMV) (1), Turnip mosaic virus (TuMV), Tomato spotted wilt virus (TSWV), Broad bean wilt virus (BBWV), Tomato mosaic virus (ToMV), Pepper veinal mottle virus (PVMV), and Ageratum yellow vein virus (AYVV) (4). In May 2007 (late period of growing season) in central Taiwan, systemic necrotic spots, which are similar to that caused by LNV (2), were found on approximately 20% of the lisianthus plants. Spherical virus particles, approximately 32 nm in diameter, were found in the crude sap of infected lisianthus collected from the fields. However, the diseased samples did not react with antisera against domestic lisianthus-infecting spherical viruses, LNV (2) and CMV (1). A virus culture was isolated via mechanical inoculation on Chenopodium quinoa and serologically identified as Carnation mottle virus (CarMV) by ELISA, western blotting, and immunoelectron microscopy using antiserum against the CarMV zantedeschia strain (3). The virus induced necrotic local lesions on the inoculated leaves of C. quinoa, C. amaranticolor, Gomphrena globosa, Cucurbita moschata, Phaseolus angularis, P. vulgaris, and Vigna unguiculata. Lisianthus was previously reported as a local lesion host for CarMV (3). In current studies with 8 of 10 lisianthus plants, the newly isolated virus induced necrotic local lesions on inoculated leaves 20 days post inoculation (dpi). However, systemic necrotic lesions on noninoculated upper leaves, as were observed in the fields, appeared 120 dpi on inoculated plants, indicating that CarMV induces systemic infection in lisianthus during late growth stages. Noninoculated plants did not develop symptoms. Complementary DNA fragments of viral genomic RNA were amplified with a specific primer of the coat protein gene (3) and sets of degenerate primer for CarMV. The amplified cDNA fragments were cloned and sequenced. The full-length sequence was submitted as GenBank Accession No. FJ843021. The genomic RNA consists of 4,003 nucleotides and has an identical genome organization to that reported for members of the genus Carmovirus. The nucleotide sequence of the full-length genome shares more than 95% identity to isolates of CarMV (GenBank Accession Nos. AF192772, AJ304989, AJ811998, NC_001265, and X02986), and the nucleotide and deduced amino acid sequence of coat protein shares more than 98% identity with that of CarMV-TW (AY383566) (3), CarMV-FO25 (EF622206), CarMV-Italy-Ca1 (EF622207), and CarMV-Netherland Ca2 (EF622210). To our knowledge, this is the first report of natural infection of CarMV in lisianthus in Taiwan. References: (1) C. C. Chen and C. C. Hu, Plant Prot. Bull. 41:179, 1999. (2) C. C. Chen et al. Plant Dis. 84:506, 2000. (3) C. C. Chen et al. Plant Dis. 87:1539, 2003. (4) Y. H. Cheng et al. J. Taiwan Agric. Res. 58:196, 2009.

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Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses

10.32747/2000.7575269.bard ◽

2000 ◽

Author(s):

Ron Gafny ◽

A.L.N. Rao ◽

Edna Tanne

Keyword(s):

Coat Protein ◽

Fusion Protein ◽

Complex Disease ◽

Movement Protein ◽

Full Length ◽

35S Promoter ◽

Cdna Clones ◽

Grapevine Virus ◽

Grapevine Virus A

Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.

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Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2005 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2005-2013 ◽

Cited By ~ 20

Author(s):

L E Hann ◽

A C Webb ◽

J M Cai ◽

L Gehrke

Keyword(s):

Protein Synthesis ◽

Coat Protein ◽

Mosaic Virus ◽

Untranslated Region ◽

Alfalfa Mosaic Virus ◽

Full Length ◽

Site Directed Mutagenesis ◽

Xenopus Laevis Oocytes ◽

Virus Coat Protein ◽

Virus Coat

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.

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Efficient chimeric promoters derived from full-length and sub-genomic transcript promoters of Figwort mosaic virus (FMV)

Journal of Biotechnology ◽

10.1016/j.jbiotec.2011.01.015 ◽

2011 ◽

Vol 152 (1-2) ◽

pp. 58-62 ◽

Cited By ~ 18

Author(s):

Rajiv Ranjan ◽

Sunita Patro ◽

Sangeeta Kumari ◽

Deepak Kumar ◽

Nrisingha Dey ◽

...

Keyword(s):

Mosaic Virus ◽

Full Length ◽

Figwort Mosaic Virus ◽

Chimeric Promoters

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PacBio full length transcript sequencing and Illumina transcriptome insight into immune defense mechanism of Litopenaeus vannamei under ammonia-N stress

Aquaculture ◽

10.1016/j.aquaculture.2021.736457 ◽

2021 ◽

Vol 536 ◽

pp. 736457

Author(s):

Xin Zhang ◽

Luqing Pan ◽

Ruixue Tong ◽

Yufen Li ◽

Yimeng Tian ◽

...

Keyword(s):

Litopenaeus Vannamei ◽

Defense Mechanism ◽

Immune Defense ◽

Full Length ◽

Full Length Transcript ◽

Insight Into

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Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus

Archives of Virology ◽

10.1007/s00705-021-05018-w ◽

2021 ◽

Vol 166 (5) ◽

pp. 1427-1431

Author(s):

Buyang Chen ◽

Qi Lin ◽

Yueyan Yin ◽

Liangliang Jiang ◽

Fang Wang ◽

...

Keyword(s):

Mosaic Virus ◽

Cdna Clone ◽

Full Length ◽

Full Length Cdna ◽

Aucuba Mosaic

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Multiple aromatic amino acids are involved in potyvirus movement by forming π-stackings to maintain coat protein accumulation

Phytopathology Research ◽

10.1186/s42483-021-00088-9 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Zhi-Yong Yan ◽

Xiao-Jie Xu ◽

Le Fang ◽

Chao Geng ◽

Yan-Ping Tian ◽

...

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Infectious Clone ◽

Three Dimensional ◽

Cell Movement ◽

Systemic Infection ◽

Watermelon Mosaic Virus ◽

Protein Accumulation ◽

Three Dimensional Model ◽

Aromatic Residues

AbstractCoat protein (CP) is required for potyviruses to move and establish a systemic infection in plants. π-stackings formed by aromatic residues play critical roles in maintaining protein stability and functions. As we know, many aromatic residues located in the core region of potyvirus CPs are conserved. However, their roles in potyvirus infection remain largely unknown. Here, through analysis of the three-dimensional model of the tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) CP, 16 aromatic residues were predicated to form π-stackings. The results of transient expression experiments demonstrated that deletion of any of these 16 aromatic residues reduced CP accumulation. Infectivity assays showed that deletion of any of these aromatic residues in the TVBMV infectious clone abolished cell-to-cell movement and reduced replication of the virus. Substitution of Y105 and Y147 individually with non-aromatic residues alanine or glycine reduced CP accumulation, virus replication, and abolished the ability of TVBMV to move intercellularly, while substitution of these two residues individually with aromatic residues phenylalanine or tryptophan, had no or little effect on CP accumulation and TVBMV systemic movement and replication. Similar results were obtained from the CP mutants of watermelon mosaic virus (WMV, genus Potyvirus). Taken together, our results demonstrate that multiple aromatic residues in CP are involved in potyvirus movement by forming π-stackings to maintain CP accumulation.

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