Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot.

Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 µs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 µs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.

Model System-Guided Protein Interaction Mapping for Virus Isolated from Phloem Tissue.

Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host-virus protein interaction studies. In this study, Potato leafroll virus (PLRV)-host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation (co-IP) support matrices coupled to mass spectrometry (MS), we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact in complex with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host Nicotiana benthamiana. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation, and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.

Compositional equivalency of RNAi-mediated virus-resistant transgenic soybean and its nontransgenic counterpart.

RNA silencing or RNA interference (RNAi), which is triggered by double-stranded RNA (dsRNA), is an evolutionarily conserved process that is active in a wide variety of eukaryotic organisms. Engineering plants with hairpin construct in which the viral gene is arranged in inverted repeats (IR) renders plants resistant to plant virus infection. However, there is no report on whether biologically important changes occurred by the insertion of IR, which confer transgenic plants virus resistance. In the present study, the compositions of virus-resistant transgenic soybean seeds developed by insertion of three short IRs, each containing the specific, highly conserved sequences derived from one virus, were compared with those of nontransgenic counterparts by applying the principle of substantial equivalence to determine whether significant undesirable biological changes occurred by IR insertion. The results revealed that the nutrient components as well as antinutrient contents of these virus-resistant soybean lines are substantially equivalent to those of the nontransgenic counterparts, and the majority of the measured amounts of nutritional components and antinutrient contents are well within the range of values reported for other commercial soybean lines. The results imply that no biologically important changes occurred by the insertion of IRs in the RNAi-mediated virus-resistant transgenic soybeans. The results can serve as baseline information for developing RNAi-mediated transgenic soybean cultivars or other crops with broader spectrum virus resistance.

Tertiary structural and functional analyses of a viroid RNA motif by isostericity matrix and mutagenesis reveal its essential role in replication.

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.

[Molecular identity of double-stranded RNA-elements of viral nature isolated from the sugar beet].

Hybridization analysis of the double-strand RNA-elements isolated from the sugar beet of the Kyiv, Vinnytsya and Poltava Regions was carried out. A positive signal has been obtained with the dsRNA fragment, 1.9 kbp long, which codes the viral RNA- dependent RNA-polymerase having high level of homology with the analogous enzyme of mycoviruses from Partitiviridae family. Molecular identity of these dsRNA-elements in the sugar beet samples from the Vinnytsya and Poltava regions has been shown The RT-PCR test system has been developed for detecting the nondescribed mycovirus which is proposed to be called Helicobasidium purpureum partitivirus.

Sequence divergence of four soilborne sugarbeet-infecting viruses.

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4-99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.

Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein.

The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants. Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein. Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-alpha. Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B. The nuclear export signal (NES) was characterized by mutagenesis. A GFP-p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells. The symptom phenotype induced by expression of GFP-p25 during infection was similar to that induced by wild-type virus. Studies with mutated derivatives of GFP-p25 revealed that symptom phenotype was altered when the subcellular localization of GFP-p25 was modified.

Structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by NMR and thermodynamic analysis.

The 69 nucleotide left-terminal domain (T(L)) of the potato spindle tuber RNA viroid (PSTVd) constitutes one of its five structural elements. Due to a twofold complementary sequence repeat, two possible conformations are proposed for the T(L) secondary structure; an elongated-rod and a bifurcated form. In the present study, two T(L) mutants were designed that remove the symmetry of the sequence repeats and ensure that either the bifurcated or the elongated-rod conformation is thermodynamically favored. Imino 1H and 15N resonances were assigned for both mutants and the native T(L) domain based on 1H-1H NOESY and heteronuclear 1H-15N HSQC high-resolution NMR spectra. The NMR secondary structure analysis of all constructs establishes unambiguously the elongated-rod form as the secondary structure of the native T(L) domain. Temperature-gradient gel electrophoresis and UV melting experiments corroborate these results. A combined secondary structure and sequence analysis of T(L) domains of other Pospiviroidae family members indicates that the elongated-rod form is thermodynamically favored for the vast majority of these viroids.

Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins.

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

Analysis of a three-dimensional structure of Potato leafroll virus coat protein obtained by homology modeling.

Viruses of the family Luteoviridae are ssRNA plant viruses that have particles that exhibit icosahedral symmetry. To identify the residues that might be exposed on the surface of the Potato leafroll virus (PLRV; genus Polerovirus, family Luteoviridae) capsid, and therefore involved in biological interactions, we performed a structural analysis of the PLRV coat protein (CP) on the basis of comparisons with protein sequences and known crystal structures of CPs of other viruses. The CP of PLRV displays 33% sequence similarity with that of Rice yellow mottle virus (genus Sobemovirus) when the sequences were aligned by using the hidden Markov model method. A structure model for PLRV CP was designed by protein homology modeling, using the crystal structure of RYMV as a template. The resulting model is consistent with immunological and site-directed mutagenesis data previously reported. On the basis of this model it is possible to predict some surface properties of the PLRV CP and also speculate about the structural evolution of small icosahedral viruses.

The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants.

Molecular data on Potato mop-top virus (PMTV), genus Pomovirus, is currently mostly based on analysis of two Scottish isolates, PMTV-S and PMTV-T. Here we report the complete sequence of "the coat protein (CP) encoding RNA" of an isolate of PMTV obtained from the field in Sweden. Our data show that this RNA (3134 nt) is the second largest of the three RNA species in the tripartite PMTV genome, and it should, therefore, be referred to as RNA 2. This nomenclature is consistent with other pomoviruses. The sequence of the readthrough domain (RT) of RNA 2 was determined also in two additional field isolates of PMTV from Finland and Denmark. All three isolates contained a novel, 109 nucleotides long sequence at the 3'-end of the RT, which has not been found in PMTV-S and PMTV-T. Hence, our data suggest that the RNA 2 sequences previously described for the isolates PMTV-T and PMTV-S may represent deletion derivatives. The C-proximal half of RT contained many amino acid (aa) differences among the isolates, in contrast to only few aa differences in the N-proximal part of RT. Deletion variants of RNA 2 were generated from the Nordic isolates in potato tubers infected in the field, and in the mechanically inoculated test plants. All deletions started within a short region (18 nt) and removed 558-940 nt from the 3'-end of RT region. This study for the first time describes the full-length sequence of the "CP-encoding RNA" (RNA2) of PMTV, and reveals considerable aa variability and occurrence of deletion variants of RT in the field isolates of PMTV.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Refined structure of desmodium yellow mottle tymovirus at 2.7 A resolution.

Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.

Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterranea.

A monofungal culture of Spongospora subterranea was unable to acquire and transmit the T isolate of potato mop-top pomovirus (PMTV-T), which has been maintained by manual transmission in the laboratory for 30 years. A recently obtained field isolate (PMTV-S) was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the readthrough (RT) protein-coding region of PMTV-S showed the presence of an additional 543 nt in the 3' half of the coding region relative to that of PMTV-T. These additional nucleotides preserved the reading frame of the RT protein and inserted 181 amino acids into the RT protein. This was confirmed by a comparison by immunoblotting of the sizes of the RT protein of PMTV-T and other recent isolates of PMTV.

Correlation between bending of the VM region and pathogenicity of different Potato Spindle Tuber Viroid strains.

Only 40 of the 359 nucleotides of Potato Spindle Tuber Viroid (PSTVd) represent the virulence-modulating (VM) region. Minor sequence variations in this domain distinguish mild from severe and even necrotic strains. Our recent hypothesis (Owens RA et al., 1996, Virology 222:144-158) that these differences result in varying degrees of bending of this part of the molecule could be tested experimentally. By in vitro transcription and partial double-strand formation, three types of model RNAs were prepared and subjected to electrophoresis in polyacrylamide gels: (1) Fragments representing the VM regions of six different PSTVd strains; (2) control fragments containing a bulge-loop as a rigid bend or an internal loop as a point of increased flexibility; and (3) dsRNAs of 36, 39, and 43 bp as length standards. Migration anomalies in gels of increasing percentage were evaluated and resulted in the following conclusions. In the absence of Mg2+, the VM regions differ only in terms of flexibility. Addition of Mg2+ induces conformational changes in these RNAs. All strains but Mild exhibit a rigid bend, and the angle of bending increases monotonically with the pathogenicity of the strain. The data are discussed in terms of a mechanism of pathogenicity, that protein-binding to the VM region is the primary pathogenic event.

The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Detection of potato mop-top virus capsid readthrough protein in virus particles.

Potato mop-top furovirus (PMTV) RNA 3 encodes the 20 kDa coat protein and a larger readthrough protein of 67 kDa. The readthrough protein is expressed by suppression of the amber stop codon which terminates the coat protein gene. A 21 kDa C-terminal fragment of the readthrough protein was doned, fused to glutathione S-transferase and expressed in E. coli. An antiserum prepared against purified fusion protein was used in ELISA to detect the readthrough protein in extracts of PMTV-infected leaves. Immunogold labelling studies showed that the readthrough protein was located near one extremity of some of the virus particles.

Sequence analysis of pothos latent virus genomic RNA.

The complete genomic sequence of pothos latent virus (PoLV) has been determined. The genome organization is very similar to that of tombusviruses. The genome is 4415 nucleotides long and contains five ORFs. The 5' ORF (ORF 1) encodes a protein with a predicted molecular mass of 25 kDa and readthrough of its amber stop codon results in an 84 kDa protein (ORF 2). ORF 3 encodes the 40 kDa capsid protein. Two nested ORFs (ORFs 4 and 5) in different reading frames encode 27 and 14 kDa proteins, respectively. Amino acid sequence alignments revealed significant similarities between the readthrough portion of ORF 2 and the coat protein (ORF 3) of PoLV and the corresponding proteins of several tombusviruses. Conversely, the predicted products of ORFs 1, 4 and 5 showed only limited similarity to the equivalent tombusvirus proteins. These results support the conclusion that PoLV is a new but atypical member of the family Tombusviridae.

The nucleotide sequence of cowpea mottle virus and its assignment to the genus Carmovirus.

The genome of cowpea mottle virus (CPMoV) is a positive ssRNA of 4029 nucleotides with six major open reading frames (ORFs). A non-coding region of 34 nucleotides precedes the first AUG. ORF1 encodes a 25 kDa polypeptide of unknown function and ORF2 encodes a 56 kDa putative RNA replicase. Like other members of carmoviruses, suppression of the amber termination codon of ORF1 would produce a readthrough polypeptide of 83 kDa. ORF3 and ORF4 encode two small proteins of 7.8 and 9.8 kDa, respectively. ORF5 encodes the 40 kDa capsid protein. ORF6 is located within ORF5 but is in a different frame and has no postulated function. CPMoV RNA is blocked at the 5' end and is not polyadenylated at the 3' end. Comparison of the physicochemical properties, genomic arrangement, and predicted amino acid sequences with those of other viruses justify the assignment of CPMoV to the genus Carmovirus, family Tombusviridae.