Potyvirus Coat Protein Research Articles

Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene.

Transgenic plums containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV. Infection was monitored by evaluating symptoms, ELISA, and IC-RT-PCR. Transgenic clone C5 was highly resistant to PPV during four years of testing and displayed characteristics typical of post-transcriptional gene silencing (PTGS), including a high level of transgene transcription in the nucleus, low levels of transgene mRNA in the cytoplasm, a complex multicopy transgene insertion with aberrant copies, and methylation of the silenced PPV-CP transgene. The PPV-CP transgene was also methylated in seedlings of C5 and these seedlings were resistant to PPV. Our results show, for the first time, that PTGS functions as a mechanism for virus resistance in a woody perennial species.

660 Production of Therapeutic Proteins in Transgenic Plants and Viral Vectors

Transgenic plants and plant viruses have potential advantages over other production systems for therapeutic proteins. 1) Plants are not susceptible to human and animal pathogens, such as viruses that can contaminate mammalian and avian cell lines used for production of many vaccines. Recent experiences of “mad cow” disease and theories of the possible origin of HIV from monkey cell lines have highlighted the need for increased product safety. 2) There are established protocols for preparing naturally occurring pharmaceuticals from plants. 3) Unlike bacteria, plants recognize the same glycosylation signals as other eukaryotic expression systems such as mammalian, insect, or yeast cell cultures and can thus produce glycosylated proteins. Although there are differences between plants and other eukaryotes in the types of sugar residues added to glycosylated proteins, it has been demonstrated several times that plant-produced proteins have similar stability and bioequivalence of function and that antigenicity is similar. 4) Plants can produce high yields; a single transgenic plant could yield as much human glucocerebrosidase as 500 placentae. We expressed an epitope from HIV-1 on the surface of bean yellow mosaic potyvirus (BYMV) coat protein (CP); protein produced in transgenic plants is recognized by a human monoclonal antibody that neutralizes most HIV-1 isolates. Epitope-modified BYMV-CP can be recovered from transgenic plants by incorporation into BYMV virions following infection of the transgenic plants. Modified virions display the HIV-1 epitope in a semi-regular array that should stimulate the immune system to a greater degree than free subunits. HIV epitope-bearing BYMV has been used to immunize mice to assess the immune response.

Complementation of the Movement-Deficient Mutations in Potato Virus X: Potyvirus Coat Protein Mediates Cell-to-Cell Trafficking of C-Terminal Truncation but Not Deletion Mutant of Potexvirus Coat Protein

The cell-to-cell movement of the GUS-tagged potato virus X (PVX) coat protein (CP) movement-deficient mutant was restored by potyviral CPs of potato virus A (PVA) and potato virus Y (PVY) in Nicotiana benthamiana leaves in transient cobombardment experiments. Viral cell-to-cell movement of PVX CP mutant was complemented in Nicotiana tabacum cv. SR1 transgenic plants expressing PVY CP: PVX RNA and polymerase were detected in the PVX CP mutant-inoculated leaves of transgenic plants. These findings demonstrated the ability of the PVX CP-deficient mutant to move from cell to cell but not long distances in the transgenic plants and suggest that CPs of potex- and potyviruses display complementary activities in the movement process. Potyviral CP alone is not able to carry out these activities, since the mutated PVX CP is indispensable for restored movement. No trans-encapsidation between potyviral CP and PVX RNA was observed. Therefore, potyviral CP facilitates the PVX CP mutant movement by the mechanism that cannot be explained by coat protein substitution. Our data also suggest that CP functioning in cell-to-cell movement is not restricted to a simple passive role in forming virions.

First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania.

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5' and 3' parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with "universal" Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation.

The nucleotide sequence of the 3′-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production

A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA.

Characterization of a monoclonal antibody recognizing a DAG-containing epitope conserved in aphid transmissible potyviruses: evidence that the DAG motif is in a defined conformation

Two viral proteins, the helper component-protease and the coat protein, are required for the non-persistent aphid transmission of potyviruses. In the potyvirus coat protein, the tripeptide aspartate–alanine–glycine (DAG) has often been shown to be involved. A monoclonal antibody, raised against a synthetic decapeptide containing the DAG tripeptide, reacted with the peptide as well as with isolates of soybean mosaic, tobacco etch and tobacco vein mottling potyviruses. Experiments indicate that the monoclonal antibody recognizes a conformational rather than a sequential epitope. The data support the suggestion that the DAG region plays a structural role to determine a coat protein-helper component-protease conformation that influences aphid transmission.

Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus.

Antisera to the bacterially expressed nonstructural proteins (NSP) HC-Pro, CI, NIa, and NIb and the coat protein (CP) of plum pox potyvirus (PPV) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. The antisera reacted with NSP and CP of PPV on immunogold-labelled ultrathin sections. Antiserum to CP reacted with virions of seven out of 18 other potyviruses. CP was distributed throughout the cytoplasm of infected cells. Antisera to PPV NSP specifically reacted with virus-specific cytoplasmic and/or nuclear inclusions induced by 17 different potyviruses. NSP were furthermore localized in confined cytoplasmic areas in between complex accumulations of virus-specific inclusions. Cylindrical inclusions induced by the potyviruses were proven to consist of CI protein. Most other cytoplasmic or nuclear inclusions were shown to be composed of two or more NSP. An unexpected composition of virus-induced inclusions was observed for the crystalline nuclear inclusions of tobacco etch virus. Here, in addition to the expected presence of NIa and NIb, HC-Pro could be demonstrated. Furthermore, amorphous cytoplasmic inclusions induced by papaya ringspot virus contained the expected HC-Pro but additionally NIa, NIb and CI. Beet mosaic virus-induced nuclear inclusions ('satellite bodies') contained in their electron-dense matrix NIa, NIb, Hc-Pro and CI and in their lacunae CP in bundles of virion-like filaments. The results indicate that all cytoplasmic or nuclear inclusions of potyviruses have to be regarded as deposition sites of excessively produced viral NSP.

Expression of the potyvirus coat protein mediated by recombinant vaccinia virus and assembly of potyvirus-like particles in mammalian cells.

The coat protein of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed using a recombinant vaccinia virus (VV) system. Ultra-thin section electron microscopy demonstrated that the coat protein assembled into potyvirus-like particles (PVLPs) in recombinant VV infected cells. Infection of cells with two additional VV recombinants expressing coat protein plus N-terminal and N- and C-terminal extensions also resulted in the formation of PVLPs. These results suggest that the ability of VV to express the potyvirus coat protein at sufficient levels to allow PVLP formation in vitro, could make VV a suitable vector for the delivery of PVLPs displaying vaccine antigens in vivo without the need for particle purification and/or inclusion of adjuvant. Use of such a vaccine strategy would also benefit from the proven advantages of poxviruses as vaccines such as stability in a freeze dried form, resistance to environmental factors and the potential for oral administration.

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

Differentiation Among Potyviruses Infecting Sweet Potato Based on Genus-and Virus-Specific Reverse Transcription Polymerase Chain Reaction.

Knowledge of virus diseases affecting sweet potato has been complicated due to the frequent occurrence of mixed infections and difficulties in isolating and purifying sweet potato viruses. A combined assay of reverse transcription and polymerase chain reaction (PCR) utilizing degenerate genus-specific primers POT1 and POT2 was applied to 18 sweet potato clones from China. The primers were designed to amplify the variable 5' terminal region of the potyvirus coat protein gene. Molecular analysis of the amplified fragments identified the Chinese strains of sweet potato feathery mottle virus (SPFMV-CH), sweet potato latent virus (SPLV-CH), and sweet potato virus G (SPVG-CH). Among the detected potyviruses, a distantly related strain of SPFMV-CH, tentatively named SPFMV-CH2, was identified in sweet potatoes from China. On the basis of sequence identity, SPFMV-CH2 was closely related to the common (-C) strain of that virus. Identification of a closely related strain of SPVG-CH in one sweet potato clone from China further illustrated the usefulness of broad-spectrum PCR for detecting uncharacterized viruses. The acquisition of sequence information permitted the design of virus-specific primers for detecting and differentiating SPFMV, SPLV, and SPVG.

Production of strain specific antibodies against a synthetic polypeptide corresponding to the N-terminal region of the plum pox potyvirus coat protein

Comparison of the predicted coat protein amino acid sequence of the `sweet cherry' strain of plum pox potyvirus (PPV-SwC) with the corresponding regions of several other PPV strains indicated that the main differences are in the N-terminal region. Polyclonal antibodies were produced against a synthetic peptide corresponding to the 1–14 sequence of the N-terminal region of PPV-SwC coat protein. They specifically detected PPV-SwC in different immunochemical tests.

Encapsidation of potyviral RNA in various forms of transgene coat protein is not correlated with resistance in transgenic plants.

Transgenic plants expressing either bean yellow mosaic potyvirus or chimeric potyvirus coat protein (CP) were inoculated with various potyviruses. Antigen-coated plate, indirect enzyme-linked immunosorbent assay and immunoelectron microscopy of virus purified from transgenic plants showed that progeny virions contained from < 1% to as much as 25% transgenic CP. Different levels of transcapsidation may reflect the extent of compatibility between transgene CP and the viral CP.

Resistance of Transgenic Prunus domestica to Plum Pox Virus Infection.

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.

Characterization of epitopes on zucchini yellow mosaic potyvirus coat protein permits studies on the interactions between strains.

Monoclonal antibodies (MAbs) raised against the coat protein of zucchini yellow mosaic potyvirus (ZYMV) were characterized by epitope mapping using synthetic oligopeptides. Two mutant viruses with a mutation in the amino acid sequence important for epitope recognition in vitro were obtained by site-directed mutagenesis of a full-length cDNA of ZYMV. Two MAbs, CC11 and DD2, could distinguish specifically between these mutants in mixed infections, or after sequential inoculations of muskmelons. Sequential inoculations of the mutants and analysis with MAbs CC11 and DD2 revealed that cross-protection was established between these quasi-isogenic strains within 48 h.

Resistance to Vanilla Necrosis Potyvirus in Transgenic Nicotiana benthamiana Plants Containing the Virus Coat Protein Gene

AbstractThe full‐length vanilla necrosis potyvirus (VNV) coat protein (CP) gene was introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens‐mediated transformation. Four constructs contained either: sense (+) CP sequence, antisense (‐) CP sequence, sense CP sequence with a Kozak's consensus ATG resulting in a change in the first amino acid, or antisense CP sequence with the Kozak's modification. When mechanically inoculated with a high concentration of VNV, one of the plant lines containing the full‐length sense CP gene was highly resistant to virus infection. Plants from the resistant lines expressed the CP at a relatively low level compared to susceptible lines containing the same construct. Plants containing the other three constructs were either susceptible or showed delayed symptom expression.

Coat protein gene sequences of garlic and onion isolates of the onion yellow dwarf potyvirus (OYDV).

Partial genomic sequences from an unknown garlic potyvirus and from an onion isolate of the onion yellow dwarf potyvirus (OYDV) were obtained. Comparison of the deduced amino acid sequences showed a similarity of 88% between the respective viral coat proteins. The garlic potyvirus coat protein was expressed in E. coli cells, purified, and subjected to Western blot analysis using antibodies raised against different garlic-infecting viruses. The expression protein was consistently recognised by anti-OYDV antibodies and did not react with antibodies specific for leek yellow stripe potyvirus (LYSV), garlic common latent carlavirus (GCLV) and shallot latent carlavirus (SLV). Besides, the garlic potyvirus coat protein was obtained as a fusion protein and used as antigen to produce polyclonal antibodies. These antibodies reacted with purified OYDV virions, but failed to recognise LYSV particles. In the light of this evidence the garlic potyvirus was identified as the garlic strain of OYDV.

Sequence Analysis of the Genomic RNA 3′‐terminal Region of a Potyvirus from Brachiaria spp. Related to Gulneagrass Mosaic Virus

AbstractThe 3'terminal region of the genomic RNA of a potyvirus isolated from Brachiaria spp. which is related to guinea grass mosaic virus (GGMV), was compared to that of members of the sugarcane mosaic subgroup of poty viruses. Reverse transcription‐polymerase chain reaction (RT‐PCR) of total nucleic acid extracted from infected B. brizantha resulted in the amplification of a DNA product of c. 0.9 kb. This product was cloned and sequenced, it has an open reading frame of 149 amino acid residues, and a 3'untranslated region (UTR) of 474 nucleotides. Optimal alignments of the 3'UTRs of the Brachiaria potyvirus and members of the sugarcane mosaic subgroup of potyviruses showed that it has nucleotide sequence similarities of c. 42. 46, 46, and 46% to maize dwarf mosaic virus strain A, sugarcane mosaic virus strains SC and MDB, and sorghum mosaic virus strain SCH, respectively. It had higher nucleotide sequence similarities of c. 88, 89, and 92% with Johnsongrass mosaic virus (JGMV) strains MDO, MDKSl and JG, respectively. Analyses of the deduced amino acid sequence of the Brachiaria potyvirus coat protein fragment revealed a 97.3% similarity to the JG strain of JGMV to which it is scrologically related. We propose that the Brachiaria potyvirus now be considered a strain of JGMV (designated JGMV‐Brac).

Sequence of the 3'-terminal region of a Zimbabwe isolate of cowpea aphid-borne mosaic virus (CABMV).

The 3'-terminal 1221 nucleotides of a Zimbabwe isolate of cowpea aphid-borne mosaic potyvirus (CABMV) genome have been sequenced. The sequence comprises an open reading frame (ORF) of 990 nucleotides and a 3' non-coding-region of 231 nucleotides followed by a poly-A. The ORF has high similarity to NIb and coat proteins (CP) of potyviruses. A potential CP Q/S cleavage site was identified, yielding a CP of 30.5 kDa containing 275 amino acids. The CABMV sequence is closely related to that of South African passiflora virus (SAPV) which should therefore be regarded as a strain of CABMV.

Chimeric potyvirus-like particles as vaccine carriers.

Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.

Stability, Host Range, and Distribution of Kalanchoe Mosaic Potyvirus inKalanchoe blossfeldiana

The distribution of Kalanchoe mosaic potyvirus (KMV) coat protein in Kalanchoe blossfeldiana was found to be very uneven, both in individual leaves and among leaves from different parts of the plant. Amounts of viral coat protein were highest in well-developed leaves in the middle of the plants and lowest in the older bottom leaves and in the small top leaves just appearing. For routine detection it is recommended to test leaf pairs number 3 to 5 from the top and to include at least two repetitions per plant. There was a high variation among different cultivars of K. blossfeldiana both in expression of symptoms and in coat protein concentration as measured by enzyme-linked immunosorbent assay (ELISA). Expression of symptoms did not correlate with ELISA values either in naturally infected or in mechanically inoculated plants, and many latent infections were noted. Symptoms were most pronounced in spring coinciding with vigorous plant growth. None of 17 other Kalanchoe spp. mechanically inoculated developed symptoms even though half were latently infected. KMV was infective in a 10 -5 to 10 -6 dilution of plant sap, and remained infectious after heating to 55°C, and after storage in sap at room temperature for 16 days.