Coat proteins of two filamentous plant viruses display NTPase activity in vitro

Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity.     

Physical and Chemical Properties of Potato Aucuba Mosaic Virus and its Coat Protein

Some properties of potato aucuba mosaic virus (PAMV) were studied. PAMV virions have a buoyant density in CsCl of 1.315 g/cm³ and RNA content of 5.06 ± 0.1 %. Limited proteolysis of the PAMV capsid protein at N-termini has been found in the process of virus isolation and storage. The race of protein degradation was considerably decreased by the presence of phenylmethyl sulphonylfluoride — a protease inhibitor — in resuspending buffer. Molecular weight estimates were 27,480 ± 319 for native PAMV protein and 24,113 ± 129 and 21,634 ± 124 for main breakdown products. Amino acid composition of the native PAMV protein seems to be very similar to that of native PVX protein and differs only in threonine, leucine, histidine, tyrosine and methyonine. All studied properties of PAMV and its protein confirm the belonging of PAMV to the potexvirus group.     

The construction of a model of the envelope proteins of potex-group viruses

Amino acid sequences of coat protein of potato X-virus, aucuba potato mosaic, papaya mosaic and white clover mosaic have been comparatively studied. The results of this comparative study have become the basis for prediction of the secondary structure of the viruses and for construction of a model of potexvirus coat protein. The potexvirus coat protein was based on the three-layered arrangement of five alpha spirals. It is supposed that RNA molecule is within the protein subunit. Availability of common amino acid sequences in alpha spirals of coat proteins of TMV and potexviruses is established and this permits supposing their common origin.     

Viruses detected in Ullucus tuberosus (Basellaceae) from Peru and Bolivia

Ullucus tuberosus (Basellaceae) plants from 12 locations in the Andean highlands of Peru and Bolivia contained complexes of either three or four viruses. Specimens from six sites in Peru contained a potexvirus, a tobamovirus, a potyvirus and a comovirus, but those from another location lacked the potexvirus. All samples from five sites in Bolivia lacked the tobamovirus. The potexvirus (PMV/U) is a strain of papaya mosaic virus differing slightly from the type strain (PMV/T) in inducing milder symptoms in some common hosts and failing to infect a few other species. It symptomlessly infected U. tuberosus, and infected 15 of 29 species from seven of nine other families. PMV/U showed a close serological relationship to PMV/T and to boussingaultia mosaic virus and a distant relationship to commelina virus X, but it is apparently unrelated to any of ten other potexviruses. The tobamovirus (TMV/U) induced symptomless or inconspicuous infection in U. tuberosus, and infected 21 of 30 species from six of eight other families. It showed a very distant serological relationship to some strains of ribgrass mosaic, tobacco mosaic and tomato mosaic viruses, but failed to react with antisera to cucumber green mottle mosaic, frangipani mosaic, odontoglossum ringspot and sunn-hemp mosaic viruses. The potyvirus, tentatively designated ullucus mosaic virus (UMV), alone in U. tuberosus induced leaf symptoms indistinguishable from the chlorotic mottling and distortion found in naturally infected plants. UMV infected 12 of 20 species from four other families, and was transmitted in the non-persistent manner by Myzus persicae. It showed a distant serological relationship to only two (bidens mottle and alstroemeria mosaic) of 25 members or possible members of the potyvirus group tested. Some hosts and properties of the comovirus are described in an accompanying paper. None of the four viruses infected potato (Solanum tuberosum) and, with the possible exception of UMV, they differed from viruses reported previously to infect three other vegetatively propagated Andean crops (Oxalis tuberosa, Arracacia xanthorrhiza and Tropaeolum tuberosum).     

Conserved and variable elements in RNA genomes of potexviruses

The nucleotide sequences of genomic RNAs and predicted amino acid sequences of two strains of potato virus X and white clover mosaic potexvirus were compared to each other, and the proteins of different plus-RNA-containing plant viruses. The predicted non-virion proteins of potexviruses have direct sequence homology and common structural peculiarities with those of several 'Sindbis-like' plant viruses. The most conserved amino acid sequences were found to be located in the polypeptide encoded by the long 5'-proximal open reading frame (ORF1). The putative polypeptide encoded by the ORF2 starting beyond the ORF1 stop codon is clearly related to the presumptive NTP-binding domain of the ORF1-coded polypeptide. These results suggest possible functions for all of the potexvirus proteins and also indicate that potexviruses have a genome organization which is considerably different from that of other plant viruses.     

Virions and membrane proteins of the potato X virus interact with microtubules and enables tubulin polymerization in vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

Difference in the mechanism of self-assembly in vitro in tobacco mosaic virus and potato virus X

The effects of 254 nm UV-irradiation of tobacco mosaic virus (TMV) and potato virus X (PVX) RNA preparations on the RNA ability to self-assembly in vitro with the viral coat proteins were studied. It was found that while TMV RNA ability to assemble with the homologous protein is rapidly inactivated by the UV-irradiation, PVX RNA ability to be encapsidated by the PVX coat protein is quite resistant to the irradiation. More than that, the irradiation of TMV RNA with the dose strongly inhibiting its assembly with the homologous protein, did not result in any significant inhibition of this RNA ability to be coated with the PVX protein. The results testify to the profound differences in the mechanisms of RNA-protein interactions in the processes of self-assembly in vitro of tobamoviruses and potexviruses.     

Modified model of potato virus X coat protein structure

We propose the modified model of the structure of coat protein (CP) subunits in filamentous virions of potato virus X (PVX). The model is similar to the one proposed by us in 2001 for the CP of another helical plant virus (potato virus A) belonging to other (potyvirus) group. In this model the PVX CP molecule consist of two main domains--a bundle of four alpha-helices located close to the virion long axis and a so-called RNP-fold (or abCd-fold) located near the virion surface. Basing on this model we suggest possible mechanism of described by J.G. Atabekov and colleagues structural transition ("remodeling") of the PVX virions resulting from their interaction with virus-specific TGB-1 protein.     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.     

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.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm(-1) region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm(-1)), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.     

Genetically engineered resistance to potato virus X in four commercial potato cultivars

The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP benzyl-aminopurine - BCIP 5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt - CaMV cauliflower mosaic virus - CP capsid protein - GA₃ gibberellic acid - Kbp kilobase pair - NAA naphthalene acetic acid - NBT nitroblue tetrazolium chloride - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PMSF phenyl methyl sulfonyl fluoride - PVX potato virus X - PVY potato virus Y     

Mapping the surface-exposed regions of papaya mosaic virus nanoparticles

In general, the structure of the papaya mosaic virus (PapMV) and other members of the potexviruses is poorly understood. Production of PapMV coat proteins in a bacterial expression system and their self-assembly in vitro into nanoparticles is a very useful tool to study the structure of this virus. Using recombinant PapMV nanoparticles that are similar in shape and appearance to the plant virus, we evaluated surface-exposed regions by two different methods, immunoblot assay and chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or diethyl-pyrocarbonate followed by mass spectrometry. Three regions were targeted by the two techniques. The N- and C-termini were shown to be surfaced exposed as expected. However, the region 125-136 was revealed for the first time as the major surface-exposed region of the nanoparticles. The presence of linear peptides at the surface was finally confirmed using antibodies directed to those peptides. It is likely that region 125-136 plays a key role in the lifecycle of PapMV and other members of the potexvirus group.     

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.     

Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.     

Structural Lability of Barley Stripe Mosaic Virus Virions

Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.