Production of polyclonal antibodies to a recombinant coat protein of potato virus Y

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.     

Modified model of the structure of the potato virus X coat protein

A modified model was proposed for the tertiary structure of the coat protein (CP) molecules in potato virus X (PVX) virions, similar to the original model of 2001 describing the structure of CP of potato virus A, a member of another group of filamentous viruses. According to the new model, CP comprises two main structural domains, namely, a bundle of α-helices, located near the long axis of the virion, and the socalled RNP fold (or abCd fold), located in the vicinity of its surface. The model made it possible to suggest a possible mechanism of the PVX virion structural rearrangement (remodeling) resulting from translational activation of virions by the TGB1 movement protein according to Atabekov and colleagues.     

Use of three-hybrid system to detect RNA-binding activity of alfalfa mosaic virus coat protein

We used yeast three-hybrid system, for studying interaction of alfalfa mosaic virus coat protein AMVCP (AMVCP) with RNA4, which codes this protein. We have shown that AMVCP with high affinity is bound to plus-chain of RNA4 in vivo. The mutational analysis has shown, that the N-terminal part of AMVCP (aa 1 to 85) contains RNA-binding domain. C-terminal part of this protein (aa 86 to 221) does not participate in direct interaction with RNA4. However activity of the reporter-gene LacZ, which codes beta-galactosidase, in case of interaction only N-terminal part of AMVCP is five times lower, in comparison with full-length hybrid protein, that confirms that the tertiary structure of full-length AMVCP is more favourable for interaction with RNA4.     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A

Plant viruses encode movement proteins (MPs) to facilitate transport of their genomes from infected into neighboring healthy cells through plasmodesmata. Growing evidence suggests that specific phosphorylation events can regulate MP functions. The coat protein (CP) of potato virus A (PVA; genus Potyvirus) is a multifunctional protein involved both in virion assembly and virus movement. Labeling of PVA-infected tobacco leaves with [(33)P]orthophosphate demonstrated that PVA CP is phosphorylated in vivo. Competition assays established that PVA CP and the well characterized 30-kDa MP of tobacco mosaic virus (genus Tobamovirus) are phosphorylated in vitro by the same Ser/Thr kinase activity from tobacco leaves. This activity exhibits a strong preference for Mn(2+) over Mg(2+), can be inhibited by micromolar concentrations of Zn(2+) and Cd(2+), and is not Ca(2+)-dependent. Tryptic phosphopeptide mapping revealed that PVA CP was phosphorylated by this protein kinase activity on multiple sites. In contrast, PVA CP was not phosphorylated when packaged into virions, suggesting that the phosphorylation sites are located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in planta.     

A pH-dependent conformational change in the coat protein subunits from potato virus X.

Both the circular dichroism and fluorescence spectra of the dissociated coat protein subunits from potato virus X changed substantially over the pH range 8 to 4, irreversible changes resulted below pH 4, with tyrosyl and tryptophanyl residues affected most. The titration curves show a pKa of about 5.6 and do not require cooperative interactions between the coat protein subunits, thus they are in marked contrast to titrations of tobacco mosaic virus A-protein. The spectra of the intact virus were little changed between pH 8 and 4 and suggested that the coat protein was locked into a conformation similar to that of the subunits in solution at pH 7. It is proposed that the pH induced conformational change is responsible for determining the acidic branch of the pH profile for reconstitution of potato virus X from its dissociated coat protein subunits and RNA.     

Nucleotide sequence of the open reading frames adjacent to the coat protein cistron in potato virus X genome

The cloned cDNA copies corresponding to 1300 nucleotides adjacent to the 3'-terminal poly(A) tract of the potato virus X (PVX) genome have been sequenced. The amino acid sequences of three open reading frames were deduced from the nucleotide sequence. Two putative small nonstructural polypeptides corresponding to the open reading frames adjacent to the coat protein cistron possess some properties of membrane-associated proteins. Direct sequence homology and common structural peculiarities exist between the PVX small proteins and the putative small nonstructural proteins encoded by RNA 2 of hordeiviruses and furoviruses     

Comparative structural stability of subunits of the potato virus X coat protein in solution and in virus particles

Several optical methods and differential scanning calorimetry were used to study the structure and stability of free coat protein (CP) molecules and CP molecules in the virion of the potato virus X (PVX), a filamentous plant virus. All criteria suggest that PVX CP (hereinafter, CP) subunits in solution at room temperature display a certain preserved tertiary structure; however, this structure is very unstable and already denatures at 35°C. Very low concentrations of sodium dodecylsulfate or cetyltrimethylammonium bromide also disrupt the CP tertiary structure, three-five molecules of these detergents per one protein molecule being sufficient. However, the secondary structure of CP molecules does not change under the same conditions. Once included into the virion, CP subunits become considerably more stable towards increased temperature and detergents. This combination of a highly labile tertiary structure and a fairly stable secondary structure of free CP can be a structural basis for the recently discovered ability of PVX CP to assume two distinct functional states within the virion.     

Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of plants, including staple food crops. Previous cryo-electron microscopy studies of virus-like particles show that luteovirid viral capsids are built from a structural coat protein that organizes with T = 3 icosahedral symmetry. Here, we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.80-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation and suggest that the CP N-terminus and its interactions with RNA play an important role in generating capsid curvature.     

A search for evidence of virus/transgene interactions in potatoes transformed with the potato leafroll virus replicase and coat protein genes

A search was conducted to detect evidence for interactions between potato leafroll virus (PLRV)-derived transgenes expressed in Russet Burbank potato and viruses to which the transgenic plants were exposed and by which they were infected. More than 25000 plants in 442 lines transformed with 16 different coat protein gene (CP) constructs and nearly 40000 plants in 512 lines transformed with seven different replicase gene (Rep) constructs of PLRV were exposed to field infection over a 6-year period. These plants were individually inspected for type and severity of virus symptoms. Heterologous viruses found infecting the plants were identified and examined for alterations in transmission characteristics, serological affinity, host range, and symptoms. Selected isolates of PLRV from field-infected plants were examined for unusual symptoms produced in diagnostic hosts and for alteration in sedimentation properties in density gradient tubes. Viruses that were propagated in selected transgenic lines in a greenhouse were examined for similar alterations. Transmission characteristics and serological properties were not altered when they replicated in potatoes containing CP constructs in the field or greenhouse. Potato plants expressing CP or Rep constructs of PLRV were not infected in the field or in the greenhouse with viruses that do not normally infect potato. New viruses or viruses with altered sedimentation characteristics, symptoms, or host range were not detected in field-exposed or greenhouse-inoculated potato plants expressing CP or Rep gene constructs of PLRV.     

The positive correlation between dN/dS and dS in mammals is due to runs of adjacent substitutions

A positive correlation between ω, the ratio of the nonsynonymous and synonymous substitution rates, and dS, the synonymous substitution rate has recently been reported. This correlation is unexpected under simple evolutionary models. Here, we investigate two explanations for this correlation: first, whether it is a consequence of a statistical bias in the estimation of ω and second, whether it is due to substitutions at adjacent sites. Using simulations, we show that estimates of ω are biased when levels of divergence are low. This is true using the methods of Yang and Nielsen, Nei and Gojobori, and Muse and Gaut. Although the bias could generate a positive correlation between ω and dS, we show that it is unlikely to be the main determinant. Instead we show that the correlation is reduced when genes that are high quality in sequence, annotation, and alignment are used. The remaining--likely genuine--positive correlation appears to be due to adjacent tandem substitutions; single substitutions, though far more numerous, do not contribute to the correlation. Genuine adjacent substitutions may be due to mutation or selection.     

Comparative study of structural stabylity of potato virus X coat protein molecules in solution and in the virus particles

With help of several optical methods and differential scanning calorimetry we studied the structure and stability of molecules of coat protein (CP) of filamentous of potato virus X (PVX) in free state and in the virions. According to the results of all these methods, at room temperature (25 degrees C) free PVX CP subunits possess some fixed tertiary structure but this structure is highly unstable and is completely disrupted at temperatures as low as 35 degrees C. The free PVX CP tertiary structure was also disrupted by very low sodium dodecylsulfate and cetyltrimetylammonium bromide concentrations: 3 to 5 moleculs of the surfactants per the CP molecule were sufficient to induce its total disruption. At the same time, these treatments did not result in any changes in the PVX CP secondary structure. Incorporation of the CP subunits into the PVX virions resulted in a strong increase in their stability to effects of increased temperatures and surfactants. This combination of highly labile tertiary structure and rather stable secondary structure of free PVX CP subunits may represent a structural basis for recently observed capacity of the PVX CP moleculs to assume two different functional states in the virion.     

Resistance to potato leaf roll virus and potato virus Y in somatic hybrids between dihaploid Solanum tuberosum and S. brevidens

Summary Many somatic fusion hybrids have been produced between a dihaploid potato Solanum tuberosum and the sexually-incompatible wild species S. brevidens using both chemical and electrical fusion techniques. S. brevidens was resistant to both potato leaf roll virus (PLRV) and potato virus Y (PVY), the viruses being either at low (PLRV) or undetectable (PVY) concentrations as determined by enzyme-linked immunosorbent assay (ELISA). The S. tuberosum parent was susceptible to both viruses. A wide range of resistance, expressed as a decrease in virus concentration to both viruses was found amongst fusion hybrids, four of which were especially resistant. The practicality of introducing virus resistance from S. brevidens into cultivated potatoes by somatic hybridisation is discussed.     

Monoclonal antibodies specific for potato mop-top virus, and some properties of the coat protein

A method was devised which gave consistent yields (1–2 mg/kg leaves) of potato mop-top furovirus (PMTV) particles. Monoclonal antibodies (MAbs) were produced and some properties of 10 of them were studied. Four MAbs readily detected PMTV isolates from six countries in Northern Europe and Japan when the isolates were trapped with polyclonal antibody; and diagnostic tests based solely on MAbs (SCR 68 to coat plates and biotin- or enzyme-labelled SCR 69 to detect trapped virus) were devised. The pattern of reactions of the MAbs in ELISA and immunoblots suggested that they react with at least five different epitopes. PMTV coat protein preparations were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Three bands of 23.9 kd, 21.5 kd and 20.5 kd were visible in silver-stained gels and all three reacted with PMTV specific MAbs. The relative amounts of the three bands varied between different virus preparations, but the 21.5 kd band was usually the most abundant. The three bands were probably not produced by anomalous behaviour in SDS-PAGE. Moreover PMTV protein was readily degraded by trypsin treatment giving a band of 20.5 kd. Therefore the results suggest that PMTV coat protein sub-units are sensitive to degradation by plant proteases. At least two degraded forms were found when purified preparations were analysed by SDS-PAGE, and the undegraded protein was estimated to be 23.9 kd. The PMTV MAbs did not react in immunoblots with SDS-treated coat protein preparations of beet necrotic yellow vein furovirus or Indian peanut clump furovirus.