Ringspot Virus Coat Protein Research Articles

Study of interaction between Papaya ringspot virus coat protein and infected Carica papaya proteins

ABSTRACT Papaya ringspot virus (PRSV) causes the Papaya ringspot disease. Virus–host interactions appear to play a significant role in the replication, pathogenesis, and infection caused by the PRSV. PRSV coat protein is likely to be involved in the processes of RNA replication, aphid transmission, and cell-to-cell movement, which is closely related to the host cell proteins resulting in a PRSV infection. To identify the host proteins that interact with the CP in vitro, immune precipitation, in-solution trypsin digestion, and LC–MS/MS were performed. Twenty-three identified proteins that interacted with the CP are involved in cellular metabolism, transcription, signal, translation, carbohydrate metabolism, protein metabolism, stress response, photosynthesis, nucleotide metabolism, respiration, and lipid metabolism processes. The search tool for interactions of chemicals (STITCH) results show that SWP (an RNA polymerase II transcription mediator) and MPPBETA (Mitochondrial processing peptidase) are involved in known plant defense mechanisms including transcription factors, cell division, hormones, stress, mitochondrial electron transferase, respiration, and proteasome. Therefore, analyzing virus–host protein interactions at a molecular level is important to build a better understanding of the virus replication mechanism and cellular responses mounted against viruses by the host defense system.

Mutagenicity test of Streptomyces Species culture filtrate using mouse lymphoma cells

Introduction: Streptomyces species culture was isolated and identified. Its filtrate was found to be able to damage Odontoglossum ringspot virus and Cymbidium mosaic virus coat protein in our previous study. It is absolutely necessary to establish its biohazard level and toxicological profile before this filtrate or its active compound can be used for immediate disinfection of the equipment and bench surface, or even technician’s skin. Aim: In this study we intend to test the mutagenicity of this culture filtrate using the mouse lymphoma assay (MLA). Material and methods: Streptomyces species was cultured for 14 days, and the medium was collected for XTT assay to determine the highest concentration of the culture filtrate that do not cause reduction in cell number. In vitro mammalian cell gene mutation test using Mouse lymphoma L5178Y TK+/- was used to evaluate the mutagenic potential of the culture filtrate in accordance with OECD (1997), test No. 476: in vitro Mammalian Cell Gene Mutation Test. Result: Our cytotoxicity assay revealed that 1.33% of the culture filtrate was the maximal concentration that would not inflict reduction in cell number. The result of mutation test showed that with 3-hour treatment and S9 metabolic activation, the culture filtrate did not show significant mutagenicity when compared to the negative controls (ANOVA and Student t-test, p<0.05). Summary: This data suggested that the culture filtrate is not mutagenic.

Comparative analysis of the metabolome and transcriptome between green and albino zones of variegated leaves from Hydrangea macrophylla ‘Maculata’ infected by hydrangea ringspot virus

In nature, many different factors cause plants to present variegated leaves. The purpose of this study was to reveal the changes in the green and albino leaves of Hydrangea macrophylla ‘Maculata’. It was found that in the albino zone, the leaves became thinner, the chloroplast structure disappeared, and a large number of leucoplasts replaced chloroplasts. In addition, the albino zone of the leaves contained almost no chlorophyll and showed no function related to transforming and utilizing light energy, and more intense oxidative stress was observed in the albino zone of the leaves than in the green zone. RNA-seq analysis showed that the chlorophyll synthesis pathway of the albino zone of leaves was blocked. Upregulated expression of the hydrangea ringspot virus (HdRSV) coat protein (CP) gene was detected in albino tissue by RT-qPCR. Finally, combined UPLC-MS/MS and RNA-seq analyses revealed metabolic changes involving multiple pathways in albino leaf tissue, centered on the TCA cycle. We hypothesize that HdRSV may alter energy metabolism in the albino zone of leaves, including increased lipid metabolism, reduced sugar metabolism, and increased synthesis of amino acids and the viral capsid protein from ribosomes.

Molecular modeling and interaction between Arabidopsis sulfite oxidase and the GW motif of Turnip crinkle virus coat protein

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.

The detection of papaya ringspot virus coat protein using an electrochemical immunosensor

Electrochemical impedance spectroscopy-based immunosensors have enormous potential as a simple, rapid, economical, and field-deployable tool for plant pathogen detection.

Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus L.), two HCRSV mutants, designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G) were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly in vitro.

Tomato ringspot virus coat protein binds to ARGONAUTE 1 and suppresses the translation repression of a reporter gene.

RNA silencing regulates plant gene expression and antiviral defenses and functions by cleaving target RNAs or repressing translation. As a counter defense, many plant viruses encode suppressor proteins that sequester small RNAs or inactivate Argonaute (AGO) proteins. All known plant virus silencing suppressor activities eventually inhibit the degradation of target mRNAs. Using a transiently expressed green fluorescent protein (GFP) reporter gene, we show that Tomato ringspot virus (ToRSV) coat protein (CP) is a suppressor of RNA silencing that enhances GFP expression but does not prevent the degradation of the GFP mRNA or the accumulation of GFP small interfering RNAs (siRNAs). Coexpression of the CP with GFP resulted in increased association of residual GFP mRNAs with polysome fractions and reduced association of GFP siRNAs with monosome fractions. AGO1 was co-immunoprecipitated with the CP and CP expression destabilized AGO1. A WG motif within the CP was critical for the enhanced GFP expression, AGO1 interaction, and AGO1 destabilization, suggesting that the ToRSV CP acts as an AGO-hook protein and competes for AGO binding with a plant cellular GW/WG protein involved in translation repression.

Allergenicity Assessment of the Papaya Ringspot Virus Coat Protein Expressed in Transgenic Rainbow Papaya

The virus-resistant, transgenic commercial papaya Rainbow and SunUp (Carica papaya L.) have been consumed locally in Hawaii and elsewhere in the mainland United States and Canada since their release to planters in Hawaii in 1998. These papaya are derived from transgenic papaya line 55-1 and carry the coat protein (CP) gene of papaya ringspot virus (PRSV). The PRSV CP was evaluated for potential allergenicity, an important component in assessing the safety of food derived from transgenic plants. The transgene PRSV CP sequence of Rainbow papaya did not exhibit greater than 35% amino acid sequence homology to known allergens, nor did it have a stretch of eight amino acids found in known allergens which are known common bioinformatic methods used for assessing similarity to allergen proteins. PRSV CP was also tested for stability in simulated gastric fluid and simulated intestinal fluid and under various heat treatments. The results showed that PRSV CP was degraded under conditions for which allergenic proteins relative to nonallergens are purported to be stable. The potential human intake of transgene-derived PRSV CP was assessed by measuring CP levels in Rainbow and SunUp along with estimating the fruit consumption rates and was compared to potential intake estimates of PRSV CP from naturally infected nontransgenic papaya. Following accepted allergenicity assessment criteria, our results show that the transgene-derived PRSV CP does not pose a risk of food allergy.

Hibiscus chlorotic ringspot virus upregulates plant sulfite oxidase transcripts and increases sulfate levels in kenaf (Hibiscus cannabinus L.)

Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is required for encapsidation and virus systemic movement. To better understand the roles of HCRSV CP in virus infection and its interactions with host proteins, a cDNA library of kenaf (Hibiscus cannabinus L.) was constructed and screened by using a yeast two-hybrid system (YTHS) to identify CP-interacting proteins. One protein identified was sulfite oxidase (SO) and the interaction was confirmed in vitro and in vivo. The interaction was found to be associated with peroxisomes by immunofluorescent labelling of peroxisomes by an anti-SKL signal peptide antibody. Our YTHS results showed that only the P and S domains of CP interacted with SO from kenaf. This is probably due to the exposure of these two domains on the outer surface of the capsid. Peroxisomes were observed to aggregate in HCRSV-infected cells, and biochemical assays of total protein from kenaf leaf extracts showed that SO activity and SO-dependent H(2)O(2)-generating activity in the HCRSV-infected leaves increased compared with that in mock-inoculated kenaf plants.

Potential uses of in vitro expressed and purified recombinant Prunus necrotic ringspot virus coat protein gene

Prunus necrotic ring spot virus (PNRSV), an ilarvirus, has a wide host range especially on important commercial crops like almond, apple, apricot, hop, peach, and rose. PNRSV coat protein gene was cloned in expression vector pGEX-2Tk (Amersham Pharmecia, USA) using E. Coli BL 21 strain competent cells. Expression conditions were standardized for maximum recovery of soluble recombinant protein. The in vitro expressed protein was purified and used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera raising. A part of the raised and purified antisera was used in enzyme conjugate preparation. It was then formulated in the form of an ELISA-based diagnostic kit for PNRSV detection. It was also compared with the commercially available kit.

Hibiscus chlorotic ringspot virus coat protein inhibits trans-acting small interfering RNA biogenesis in Arabidopsis

Many plant and animal viruses have evolved suppressor proteins to block host RNA silencing at various stages of the RNA silencing pathways. Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) is capable of suppressing the transiently expressed sense-RNA-induced post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana. Here, constitutively expressed HCRSV CP from transgenic Arabidopsis was found to be able to rescue expression of the silenced GUS transgene. The HCRSV CP-transgenic Arabidopsis (line CP6) displayed several developmental abnormalities: elongated, downward curled leaves and a lack of coordination between stamen and carpel, resulting in reduced seed set. These abnormalities are similar to those observed in mutations of the genes of Arabidopsis RNA-dependent polymerase 6 (rdr6), suppressor of gene silencing 3 (sgs3), ZIPPY (zip) and dicer-like 4 (dcl4). The accumulation of microRNA (miRNA) miR173 remained stable; however, the downstream trans-acting small interfering RNA (ta-siRNA) siR255 was greatly reduced. Real-time PCR analysis showed that expression of the ta-siRNA-targeted At4g29770, At5g18040, PPR and ARF3 genes increased significantly, especially in the inflorescences. Genetic crossing of CP6 with an amplicon-silenced line (containing a potato virus X-green fluorescent protein transgene under the control of the 35S cauliflower mosaic virus promoter) suggested that HCRSV CP probably interfered with gene silencing at a step after RDR6. The reduced accumulation of ta-siRNA might result from the interference of HCRSV CP with Dicer-like protein(s), responsible for the generation of dsRNA in ta-siRNA biogenesis.

Post-transcriptional gene silencing is involved in resistance of transgenic papayas to papaya ringspot virus

Transgenic papayas carrying the papaya ringspot virus coat protein gene were inoculated with Papaya ringspot virus. Infection was monitored by evaluating symptoms and by reverse transcription polymerase chain reaction (RT-PCR). Among eight tested transgenic lines, clone G2 was found highly resistant to virus infection during 3 years of testing. Further analysis of this clone revealed complex multicopy transgene insertion with aberrant copies. The suspected post transcriptional gene silencing was confirmed by siRNA detection. While the R0 generation of G2 transgenic papaya was found to be fully resistant to the infection, Papaya ringspot virus was able to break this resistance in subsequent generations by suppressing post-transcriptional gene silencing (PTGS).

Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein

Viruses depend heavily on host cells for replication and exploit the host translation machinery for its gene expression using various unorthodox translation mechanisms. According to the conventional scanning model, only the 5′-proximal gene in the viral RNA is accessible to the ribosomes whereas other genes are silent. In this study, we use a model plant RNA virus, Hibiscus chlorotic ringspot virus (HCRSV), to investigate various translation mechanisms involved in regulation of the expression of internal genes. The 3′-end 1.2 kb region of HCRSV genomic and subgenomic RNAs were shown to encode four polypeptides of 38, 27, 25 and 22.5 kDa. Mutagenesis studies revealed that a CUG codon ( 2570CUG) is the initiation codon for p27, the longest of the three co-C-terminal products (p27, p25 and p22.5), and translation of p25 and p22.5 was initiated at 2603AUG and 2666AUG, respectively. Translation initiation of the p27 expression at the 2570CUG codon regulates the expression of p38, the viral coat protein through a leaky scanning mechanism and mutational analysis of an upstream open reading frame (ORF) demonstrated that initiation of the p27 expression at this CUG codon (instead of an AUG) may play a role in maintaining the ratio of p27 and p38. In addition, a previously identified internal ribosome entry site was shown to control the expression of p27 and p38 in the subgenomic RNA 2.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Introduction of Odontoglossum ringspot virus Coat Protein Gene into Cymbidium niveo-marginatum mediated by Agrobacterium tumefaciens to Produce Transgenic Plants

We cloned the Odontoglossum ringspot virus (ORSV) coat protein (CP) gene from ORSV-infected Epidendrum and constructed a pHPUNOJE-1 vector by ligating this CP gene, green fluorescence protein (GFP) gene, and hygromycin resistant (Hygr) gene into the plasmid pGreenII. This chimeric vector was mediated into A. tumefaciens LBA 4404. By infecting and co-culturing the rhizomes of Cym. niveo-marginatum with A. tumefaciens LBA 4404, ORSV CP gene transgenic shoots were obtained. GFP was observed in 64% of the 81 surviving rhizomes and in 39% of 215 regenerated shoots. The presence of ORSV CP, GFP, and Hygr genes was confirmed in the transgenic shoots by using polymerase chain reaction (PCR) analysis, whereas that of ORSV CP was confirmed by enzyme-linked immunosorbent assay (ELISA).

Sequence similarity between the viral cp gene and the transgene in transgenic papayas

The Papaya ringspot virus (PRSV) coat protein transgene present in 'Rainbow' and 'SunUp' papayas disclose high sequence similarity (&gt;89%) to the cp gene from PRSV BR and TH. Despite this, both isolates are able to break down the resistance in 'Rainbow', while only the latter is able to do so in 'SunUp'. The objective of this work was to evaluate the degree of sequence similarity between the cp gene in the challenge isolate and the cp transgene in transgenic papayas resistant to PRSV. The production of a hybrid virus containing the genome backbone of PRSV HA up to the Apa I site in the NIb gene, and downstream from there, the sequence of PRSV TH was undertaken. This hybrid virus, PRSV HA/TH, was obtained and used to challenge 'Rainbow', 'SunUp', and an R2 population derived from line 63-1, all resistant to PRSV HA. PRSV HA/TH broke down the resistance in both papaya varieties and in the 63-1 population, demonstrating that sequence similarity is a major factor in the mechanism of resistance used by transgenic papayas expressing the cp gene. A comparative analysis of the cp gene present in line 55-1 and 63-1-derived transgenic plants and in PRSV HA, BR, and TH was also performed.

Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein

Complementation of movement and coat proteins of the orchid-infecting potexvirus Cymbidium mosaic virus (CymMV) and tobamovirus Odontoglossum ringspot virus (ORSV) was investigated. Nicotiana benthamiana, which is susceptible to both CymMV and ORSV, was used as a model system. Four transgenic lines, each harbouring one of the movement protein (MP) or coat protein (CP) genes of CymMV or ORSV, were constructed. The MP of CymMV consists of three overlapping open reading frames, together called the triple-gene block (TGB). CymMV and ORSV mutants, each carrying an inactivated MP or CP, were generated from the respective biologically active full-length cDNA clones. Complementation was studied by infecting transgenic plants with in vitro transcripts generated from these mutants. The cell-to-cell movement of a movement-deficient CymMV was restored in transgenic plants carrying the ORSV MP transgene. Similarly, CymMV TGB1 transgenic plants were able to rescue the cell-to-cell movement of a movement-deficient ORSV mutant. ORSV CP transgenic plants supported systemic movement of a CymMV CP-deficient mutant. However, in these plants, neither encapsidation of CymMV RNA with ORSV CP nor CymMV CP expression was detected. Long-distance movement of an ORSV CP-deficient mutant was not supported by CymMV CP. The complementation of MPs and CPs of CymMV and ORSV facilitates movement of these viruses in plants, except for long-distance movement of ORSV RNA by CymMV CP.

Detection and identification of transgenic virus resistant papaya and squash by multiplex PCR

Two separate multiplex PCR assays were developed for the detection of transgenic papaya and transgenic squash. Papaya line 55-1 contains a genetic insertion consisting of the coat protein gene of Papaya ringspot virus strain p and is resistant to infection by this virus. A multiplex PCR was developed to specifically amplify the papaya ringspot virus coat protein transgene construct and an endogenous papaya gene sequence. A third primer set was designed to amplify the β-glucuronidase gene construct, which can distinguish between the commercial and noncommercial papaya lines 55-1 and 63-1. Squash line ZW-20 contains genetic insertions of the coat protein genes from Watermelon mosaic virus II and Zucchini yellow mosaic virus and is resistant to these two viruses. Squash line CZW-3 is similar to ZW-20 but additionally confers resistance to Cucumber mosaic virus. A second multiplex assay was developed to detect and differentiate squash lines ZW-20 and CZW-3 in a single reaction.

High-density linkage mapping revealed suppression of recombination at the sex determination locus in papaya.

A high-density genetic map of papaya (Carica papaya L.) was constructed using 54 F(2) plants derived from cultivars Kapoho and SunUp with 1501 markers, including 1498 amplified fragment length polymorphism (AFLP) markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color. These markers were mapped into 12 linkage groups at a LOD score of 5.0 and recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. This map revealed severe suppression of recombination around the sex determination locus with a total of 225 markers cosegregating with sex types. The cytosine bases were highly methylated in this region on the basis of the distribution of methylation-sensitive and -insensitive markers. This high-density genetic map is essential for cloning of specific genes of interest such as the sex determination gene and for the integration of genetic and physical maps of papaya.

Producing Transgenic `Thompson Seedless' Grape (Vitis vinifera L.) Plants

Transgenic grape plants were regenerated from somatic embryos derived from leaves of in vitro-grown plants of `Thompson Seedless' grape (Vitis vinifera L.) plants. Somatic embryos were either exposed directly to engineered Agrobacterium tumefaciens or they were bombarded twice with 1-μm gold particles and then exposed to A. tumefaciens. Somatic embryos were transformed with either the lytic peptide Shiva-1 gene or the tomato ringspot virus (TomRSV) coat protein (CP) gene. After cocultivation, secondary embryos proliferated on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 40 μg·mL-1 kanamycin (kan). Transgenic embryos were identified after 3 to 5 months under selection and allowed to germinate and develop into rooted plants on woody plant medium containing 1 μm 6-benzylaminopurine, 1.5% sucrose, 0.3% activated charcoal, and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kanamycin (kan), positive β-glucuronidase (GUS) and polymerase chain-reaction (PCR) assays, and Southern analysis.