Yellow Mosaic Virus Coat Protein Research Articles

Recombinant turnip yellow mosaic virus coat protein as a potential nanocarrier.

To display a short peptide (GSRSHHHHHH) at the C-terminal end of turnip yellow mosaic virus coat protein (TYMVc) and to study its assembly into virus-like particles (TYMVcHis6 VLPs). In this study, recombinant TYMVcHis6 expressed in Escherichia coli self-assembled into VLPs of approximately 30-32nm. SDS-PAGE and Western blot analysis of protein fractions from the immobilized metal affinity chromatography (IMAC) showed that TYMVcHis6 VLPs interacted strongly with nickel ligands in IMAC column, suggesting that the fusion peptide is protruding out from the surface of VLPs. These VLPs are highly stable over a wide pH range from 3·0 to 11·0 at different temperatures. At pH 11·0, specifically, the VLPs remained intact up to 75°C. Additionally, the disassembly and reassembly of TYMVcHis6 VLPs were studied in vitro. Dynamic light scattering and transmission electron microscopy analysis revealed that TYMVcHis6 VLPs were dissociated by 7moll-1 urea and 2moll-1 guanidine hydrochloride (GdnHCl) without impairing their reassembly property. A 10-residue peptide was successfully displayed on the surface of TYMVcHis6 VLPs. This chimera demonstrated high stability under extreme thermal conditions with varying pH and was able to dissociate and reassociate into VLPs by chemical denaturants. This is the first C-terminally modified TYMVc produced in E. coli. The C-terminal tail which is exposed on the surface can be exploited as a useful site to display multiple copies of functional ligands. The ability of the chimeric VLPs to self-assemble after undergo chemical denaturation indicates its potential role to serve as a nanocarrier for use in targeted drug delivery.

Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified. [BMB Reports 2013; 46(10): 495-500]

A short motif in the N‐terminal part of the coat protein is a host‐specific determinant of systemic infectivity for two potyviruses

Although the biological variability of Watermelon mosaic virus is limited, isolates from the three main molecular groups differ in their ability to infect systemically Chenopodium quinoa. Mutations were introduced in a motif of three or five amino acids located in the N-terminal part of the coat protein, and differing in isolates from group 1 (motif: lysine-glutamic acid-alanine (Lys-Glu-Ala) or KEA, systemic on C. quinoa), group 2 (Lys-Glu-Thr or KET, not systemic on C. quinoa) and group 3 (KEKET, not systemic on C. quinoa). Mutagenesis of KEKET in an isolate from group 3 to KEA or KEKEA was sufficient to make the virus systemic on C. quinoa, whereas mutagenesis to KET had no effect. Introduction of a KEA motif in Zucchini yellow mosaic virus coat protein also resulted in systemic infection on C. quinoa. These mutations had no obvious effect on the disorder profile or potential post-translational modifications of the coat protein as determined in silico.

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.

EXPERIMENTS ON TRANSFORMATION AND IN VITRO REGENERATION OF SQUASH VARIETIES

Squash is an important species in the Cucurbitaceae family. The development of genetic transformation offers the potential of introducing valuable traits into this crop. An efficient in vitro plant regeneration system is critical for plant transformation. The regeneration ability of three Cucurbita varieties (C. pepo convar. patissoniana 'Ovari feher', C. pepo convar. giromontiina 'Black Beauty', C. maxima convar. maxima 'Nagydobosi') was tested as a first step for further transformation experiments. The cotyledon explants of the tested varieties were cultured on Murashige and Skoog medium supplemented either with indole-3-acetic-acid [IAA] (0.1-0.9 mg/L) and benzyladenine [BA] (0.6-1.2 mg/L) in twenty-five different hormone combinations. Shoot induction via organogenesis was achieved in the tested varieties. Our second objective was to develop an efficient transformation method. The cotyledon explants were cut and soaked in Agrobacterium tumefaciens strain LBA4404 suspension containing ZYMV CP gene (zucchini yellow mosaic virus coat protein gene) After co-cultivation, the explants were put on selection media, which contained the same hormones as the regeneration media, supplemented with claforan (500 mg/L) and kanamycin (100 mg/L). Leafy shoots were transferred to root induction media supplemented with kanamycin (100 mg/L) for further selection. Regenerated plantlets with roots were transferred to sterile soil. Transformation was confirmed by PCR amplification of ZYMV CP gene in transgenic plants.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus coat-protein gene in either sense or antisense orientation.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus (BYMV) coat-protein (CP) gene in either sense or antisense (AS) orientation were developed using biolistics. Four of the plants were confirmed to carry the CP gene in the sense orientation of the gene and seven plants in the AS orientation. Two of the CP plant lines and all of the AS lines showed DNA rearrangements of the transgene in addition to an intact copy of the transgene. The copy number ranged from one to nine. Of the 11 lines, eight had only one to four copies of the transgene. Transcription of the transgene occurred for three of the CP lines and five of the AS lines as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for presence of the virus. Several transgenic plant lines containing either antiviral transgene showed a lower incidence of infection (percentage of plants infected as detected by immunoelectron microscopy) than the non-transformed plants. Most of the CP- and AS-transgenic plants that did not contain BYMV 1 month after challenge were found to contain BYMV the next season. It appeared that BYMV infection was delayed in the CP- and AS-transgenic lines but that the transgenes did not prevent eventual infection of BYMV. This is the first report of developing a floral bulb crop with antiviral genes to BYMV.

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

SUMMARYTransgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full‐length CP, the core region of the CP, and full‐length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full‐length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene‐encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.

Transformation of Wheat With the Gene Encoding the Coat Protein of Barley Yellow Mosaic Virus

Wheat plants (Triticum aestivum cv. Hartog) were stably transformed with the bar gene and the gene encoding the barley yellow mosaic virus coat protein. Cultured immature wheat embryos were bombarded with tungsten particles coated with the pEmuPAT-cp construct. Fifteen regenerating 'PPT- resistant' plants were selected on medium containing phosphinothricin. Of these, 11 plants had both the bar and cp genes integrated into the wheat genome and two plants had only the bar gene. Transmission of the two genes to progeny of two independent plants was confirmed. The barley yellow mosaic virus coat protein was detected in both the parent and progeny plants; however, bar gene expression occurred only in the parent plants.

Field Evaluation of Transgenic Squash Containing Single or Multiple Virus Coat Protein Gene Constructs for Resistance to Cucumber Mosaic Virus, Watermelon Mosaic Virus 2 and Zucchini Yellow Mosaic Virus

Transgenic inbred squash lines containing various combinations of the cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV 2) or zucchini yellow mosaic virus (ZYMV) coat protein (CP) genes were produced using Agrobacterium-mediated transformation. Progeny from lines transformed with single or multiple CP gene constructs were tested for virus resistance under field conditions, and exhibited varying levels of resistance to infection by CMV, WMV 2 or ZYMV. Most transgenic lines remained nonsymptomatic throughout the growing seasons and produced marketable fruits, while other lines showed a delay in the onset of symptoms and/or a reduction in symptom severity. A few lines failed to display any level of resistance. Depending on the CP gene used, 40 to 95% of the transgenic lines containing single CP constructs of either CMV, WMV 2 or ZYMV were resistant to the virus from which the CP gene was derived. Transgenic lines transformed with a double CP construct containing the CP genes from CMV and WMV 2, designated CW, displayed high level of resistance to CMV and WMV 2. A transgenic line, designated ZW-20, which contained the CP genes from ZYMV and WMV 2 displayed excellent resistance to ZYMV and WMV 2 in that most of the plants showed complete resistance. A few plants developed localized chlorotic dots or blotches, yet fruits remained asymptomatic. Southern blot analysis revealed that the CP inserts of some resistant plants of line ZW-20 were no longer linked to the neomycin phosphotransferase II (NPT II) gene. This loss of linkage allowed the marker gene to be separated from the virus resistance trait by Mendelian segregation. Further analysis of these plants showed that they contained multiple WMV 2 inserts which were designated B and H, the latter consisting of two hybridization signals. Analysis of inoculated plants showed that plants with the H inserts were symptomless or developed only chlorotic dots, while those without the H insert developed more prominent chlorotic blotches. In addition to lines with resistance to two viruses, a line with resistance to three viruses was also identified. Transgenic line CZW-3, transformed with the triple CP construct containing the CMV, WMV 2 and ZYMV CP genes, exhibited resistance to all three viruses. These two transgenic inbred lines, ZW-20 and CZW-3, have allowed for the development of commercial squash hybrids with multiple virus resistance.

Nucleotide sequence of a Singapore isolate of zucchini yellow mosaic virus coat protein gene revealed an altered DAG motif.

A cDNA clone of zucchini yellow mosaic virus (ZYMV) RNA was mapped to the 3' terminal region. The nucleotide sequence revealed a single open reading frame of 1035 nucleotides followed by a 3' noncoding region of 215 nucleotides. The putative protease cleavage site for the release of coat protein (CP) was deduced to be between Glu-Ser (at amino acid position 66-67), which would result in a protein of 279 amino acids. This non-aphid-transmissible Singapore isolate of ZYMV showed a change of DAG to GAG triplet near the N-terminal of the CP. The CP gene was expressed as a protein fused to the beta-galactosidase in Escherichia coli and as an unfused protein in Saccharomyces cerevisiae.

Use of enzymatic cDNA amplification as a method of detection of bean yellow mosaic virus

The C-terminal region of bean yellow mosaic virus (BYMV) coat protein gene was selected for the design of oligonucleotide primers. Reverse transcription of viral RNA present in sap of virus-infected plants followed by polymerase chain reaction (RT-PCR) was performed for amplification of a 449 baise pairs fragment. These primers supported BYMV amplification, but did not support amplification on both bean common mosaic virus (BCMV) and potato virus Y (PVY) templates.

Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.

Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis. Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA. Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer. Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus. The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo

Turnip yellow mosaic virus (TYMV) possesses a monopartite single-stranded (+) sense RNA genome in which the coat protein (cp) gene is 3′ proximal and is expressed in vivo via a subgenomic RNA. Evidence is presented here that this subgenomic RNA is synthesized in vivo by internal initiation of replication on (−) RNA strands of genomic length. The double-stranded RNAs (dsRNAs) from TYMV-infected plants have been isolated, purified, and characterized. Under native conditions, no dsRNAs (replicative intermeadites and/or replicative forms) of subgenomic length corresponding to subgenomic cp RNA can be detected by ethidium bromide staining of RNA-sizing gels or by Northern blot hybridization using RNA probes. The presence of nascent subgenomic cp (+) RNA strands on the dsRNA of genomic length has been demonstrated using two different approaches: (1) Northern blot hybridization using (−) RNA probes under denaturing conditions and (2) characterization of the 5′ ends of nascent (+) RNA strands upon labeling by vaccinia virus nucleoside-2′-methyltransferase.

In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides.

A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides. This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site. SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed. Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA. Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies. The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly. The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.

Queuosine modification of the wobble base in tRNAHis influences 'in vivo' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.

Nucleotide sequence of turnip yellow mosaic virus coat protein mRNA

The primary structure of the coat protein messenger RNA of turnip yellow mosaic virus is presented. This sequence is the first complete nucleotide sequence of the coat protein messenger of a plant virus to be reported. The coding region, consisting of 567 nucleotides, is flanked by a 5′ noncoding region of 19 nucleotides (not including the initiation codon and the cap structure) and by a 3′ noncoding region of 109 nucleotides (including the termination signal). The coat protein mRNA has a base composition identical to that of the genome RNA with, in particular, the same high content in cytosine (38%). The codons that govern the incorporation of amino acids into the coat protein are nonrandomly utilized: >50% of the time the third base of the codons used is a cytosine. This pattern of codon preference is particularly marked for Leu, lie, Val, Thr and Cys.

In Vitro Synthesis of Turnip Yellow Mosaic Virus Coat Protein in a Wheat Germ Cell-Free System

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.

Primary structure of turnip yellow mosaic virus coat protein

Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze–thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70 –75 °C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length.