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.     

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.     

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.     

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.     

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.     

A surface loop of the potato leafroll virus coat protein is involved in virion assembly, systemic movement, and aphid transmission

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.     

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.     

Translational regulation of potato virus X RNA-coat protein complexes: the key role of a coat protein N-terminal peptide

The efficiency of in vitro translation of potato virus X (PVX) RNA within vRNP complexes assembled from genomic RNA and viral CP was examined. The vRNP particles contain the 5'-proximal RNA segments encapsidated by helically arranged CP head-like portions heterogeneous in length and the CP-free RNA tail. Translation of RNA is completely repressed upon incubation with PVX CP and is accompanied by vRNP particles production. By contrast, translation is activated in vRNPs in vitro assembled using two CP forms, differing in the principals of their N-terminal peptides modification. The N-terminal peptide of PVX CP represents the major phosphorylation site(s) for Thr/Ser-specific protein kinases. It was shown that: (i) CP phosphorylation results in a translational activation of vRNP; (ii) removal of N-terminal peptide from CP abolished activation and CP retains the translation repressing ability. It was suggested that substitution of Ser/Thr residues by non-phosphorylated Ala/Gly in N-terminal peptide of the mutant CP will led to a complete inhibition of vRNP translation. However, opposite results were obtained in our experiments: (i) RNA of such mutant virus (PVX-ST) was efficiently translated within the virions; (ii) RNA of a wild-type (wt) PVX also efficiently translated in mixedly assembled vRNP "wt PVX RNA + PVX-ST CP"; (iii) opposite result (repression of translation) was obtained with "mixed" vRNP (PVX-ST RNA + wtPVX CP). Therefore, the N-terminal peptide located at the surface of the particle and of the particles plays a key role in translation activation of the RNA encapsidated in vRNP and native virions.     

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.     

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]     

Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X

The coat protein (CP) of potato virus X was localized immunocytochemically in infected leaves of susceptible Nicotiana species and shown to be targeted to the central cavity of plasmodesmata in virus-infected cells. A viral deletion mutant, in which the CP gene was replaced with the gene for the green fluorescent protein (GFP), was restricted to single, inoculated cells. However, movement of the mutant virus was rescued on transgenic plants constitutively expressing the CP gene, and the CP was again targeted to plasmodesmata. The CP was not localized to plasmodesmata in uninfected transgenic plants and, in contrast to the plasmodesmata of PVX-infected cells, the plasmodesmata of the transgenic plants did not allow the passage of 10 kDa fluorescent dextrans. We propose that the CP is not involved in plasmodesmal gating per se , but is necessary for transport of the viral RNA to, and possibly through, plasmodesmata.     

Antigenic analysis of potato virus A particles and coat protein

Five monoclonal antibodies (MAbs) were prepared to particles of potato virus A (PVA), isolate B11. In immunoblots, MAbs A1D8 and A5B6 reacted only with full length molecules of PVA coat protein (CP). Pepscan tests with overlapping octapeptides representing the whole sequence of PVA CP showed that the epitope detected by MAb A5B6 is contained in its N-terminal octapeptide. MAbs A9A4, A3H4 and A6B8 reacted with CP molecules that lacked about 5 kD of sequence at their end(s) and detected epitopes at residues 52 to 62, 64 to 73 and 75 to 82 respectively, all of which lie in the protease-resistant core of the CP. The epitope which reacts with MAb A3H4 is in a region predicted to be hydrophobic and is not detected in intact virus particles, indicating it is a cryptotope. In contrast, MAbs A6B8 and A9A4 reacted with freshly purified PVA particles but more strongly with partially degraded ones. Pepscan tests with polyclonal antibodies to PVA isolate B11 identified five additional immunogenic sequences in PVA CP and showed that regions at the N-termini of the intact and core molecules are immunodominant. PVA isolate B11 was not transmitted by aphids, and its CP N-terminal octapeptide contains the sequence DAS, which is associated with aphid-non-transmissibility in other potyviruses. MAb A5B6, which detects this region, reacted strongly in ELISA with three out of four other aphid-non-transmissible PVA isolates but only weakly with three aphid-transmissible ones, suggesting that differences in N-terminal sequence may underlie most of the differences in aphid transmissibility.     

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     

The herpes simplex virus 1 RNA binding protein US11 is a virion component and associates with ribosomal 60S subunits.

The herpes simplex virus 1 US11 gene encodes a site- and conformation-specific RNA binding regulatory protein. We fused the coding sequence of this protein with that of beta-galactosidase, expressed the chimeric gene in Escherichia coli, and purified a fusion protein which binds RNA in the same way as the infected cell protein. The fusion protein was used to generate anti-US11 monoclonal antibody. Studies with this antibody showed that US11 protein is a viral structural protein estimated to be present in 600 to 1,000 copies per virion. The great majority of cytoplasmic US11 protein was found in association with the 60S subunit of infected cell ribosomes. US11 protein associates with ribosomes both late in infection at the time of its synthesis and at the time of infection after its introduction into the cytoplasm by the virion. US11 protein expressed in an uninfected cell line stably transfected with the US11 gene associates with ribosomal 60S subunits and localizes to nucleoli, suggesting that US11 protein requires no other viral functions for these associations.     

Immunochemical analysis of the synthetic peptides incorporated into the protein antigenic determinant of the potato virus X coat]

Three peptides located in the N-terminal region of the potato virus X coat protein were synthesized by hand solid phase method for epitope mapping of this protein. One of these peptides (nanopeptide) interacted with monoclonal antibodies to native virus X. On the basis of these studies it was assumed, that amino acid sequence of the potato virus X coat protein, which included lysine residue in position 19, is located on the virion surface.     

Vaccinia virus A6L encodes a virion core protein required for formation of mature virion

Vaccinia virus A6L is a previously uncharacterized gene that is conserved in all sequenced vertebrate poxviruses. Here, we constructed a recombinant vaccinia virus encoding A6 with an epitope tag and showed that A6 was expressed in infected cells after viral DNA replication and packaged in the core of the mature virion. Furthermore, we showed that A6 was essential for vaccinia virus replication by performing clustered charge-to-alanine mutagenesis on A6, which resulted in two vaccinia virus mutants (vA6L-mut1 and vA6L-mut2) that displayed a temperature-sensitive phenotype. At 31 degrees C, both mutants replicated efficiently; however, at 40 degrees C, vA6L-mut1 grew to a low titer, while vA6L-mut2 failed to replicate. The A6 protein expressed by vA6L-mut2 exhibited temperature-dependent instability. At the nonpermissive temperature, vA6L-mut2 was normal at viral gene expression and viral factory formation, but it was defective for proteolytic processing of the precursors of several major virion proteins, a defect that is characteristic of a block in virion morphogenesis. Electron microscopy further showed that the morphogenesis of vA6L-mut2 was arrested before the formation of immature virion with nucleoid and mature virion. Taken together, our data show that A6 is a virion core protein that plays an essential role in virion morphogenesis.     

Vaccinia virus entry into cells is dependent on a virion surface protein encoded by the A28L gene

The A28L gene of vaccinia virus is conserved in all poxviruses and encodes a protein that is anchored to the surface of infectious intracellular mature virions (IMV) and consequently lies beneath the additional envelope of extracellular virions. A conditional lethal recombinant vaccinia virus, vA28-HAi, with an inducible A28L gene, undergoes a single round of replication in the absence of inducer, producing IMV, as well as extracellular virions with actin tails, but fails to infect neighboring cells. We show here that purified A28-deficient IMV appeared to be indistinguishable from wild-type IMV and were competent to synthesize RNA in vitro. Nevertheless, A28-deficient virions did not induce cytopathic effects, express early genes, or initiate a productive infection. Although A28-deficient IMV bound to the surface of cells, their cores did not penetrate into the cytoplasm. An associated defect in membrane fusion was demonstrated by the failure of low pH to trigger syncytium formation when cells were infected with vA28-HAi in the absence of inducer (fusion from within) or when cells were incubated with a high multiplicity of A28-deficient virions (fusion from without). The correlation between the entry block and the inability of A28-deficient virions to mediate fusion provided compelling evidence for a relationship between these events. Because repression of A28 inhibited cell-to-cell spread, which is mediated by extracellular virions, all forms of vaccinia virus regardless of their outer coat must use a common A28-dependent mechanism of cell penetration. Furthermore, since A28 is conserved, all poxviruses are likely to penetrate cells in a similar way.     

The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus

Papaya mosaic virus (PapMV) coat protein (CP) in Escherichia coli was previously showed to self-assemble in nucleocapsid-like particles (NLPs) that were similar in shape and appearance to the native virus. We have also shown that a truncated CP missing the N-terminal 26 amino acids is monomeric and loses its ability to bind RNA. It is likely that the N-terminus of the CP is important for the interaction between the subunits in self-assembly into NLPs. In this work, through deletion and mutation analysis, we have shown that the deletion of 13 amino acids is sufficient to generate the monomeric form of the CP. Furthermore, we have shown that residue F13 is critical for self-assembly of the CP subunits into NLPs. The replacement of F13 with hydrophobic residues (L or Y) generated mutated forms of the CP that were able to self-assemble into NLPs. However, the replacement of F13 by A, G, R, E or S was detrimental to the self-assembly of the protein into NLPs. We concluded that a hydrophobic interaction at the N-terminus is important to ensure self-assembly of the protein into NLPs. We also discuss the importance of F13 for assembly of other members of the potexvirus family.     

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.