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.

Comparison of a compatible and an incompatible pepper-tobamovirus interaction by biochemical and non-invasive techniques: chlorophyll a fluorescence, isothermal calorimetry and FT-Raman spectroscopy.

Leaves of a pepper cultivar harboring the L(3) resistance gene were inoculated with Obuda pepper virus (ObPV), which led to the appearance of hypersensitive necrotic lesions approx. 72 h post-inoculation (hpi) (incompatible interaction), or with Pepper mild mottle virus (PMMoV) that caused no visible symptoms on the inoculated leaves (compatible interaction). ObPV inoculation of leaves resulted in ion leakage already 18 hpi, up-regulation of a pepper carotenoid cleavage dioxygenase (CCD) gene from 24 hpi, heat emission and declining chlorophyll a content from 48 hpi, and partial desiccation from 72 hpi. After the appearance of necrotic lesions a strong inhibition of photochemical energy conversion was observed, which led to photochemically inactive leaf areas 96 hpi. However, leaf tissues adjacent to these inactive areas showed elevated ΦPSII and Fv/Fm values proving the advantage of chlorophyll a imaging technique. PMMoV inoculation also led to a significant rise of ion leakage and heat emission, to the up-regulation of the pepper CCD gene as well as to decreased PSII efficiency, but these responses were much weaker than in the case of ObPV inoculation. Chlorophyll b and total carotenoid contents as measured by spectrophotometric methods were not significantly influenced by any virus inoculations when these pigment contents were calculated on leaf surface basis. On the other hand, near-infrared FT-Raman spectroscopy showed an increase of carotenoid content in ObPV-inoculated leaves suggesting that the two techniques detect different sets of compounds.

Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus, a unique tobamovirus that contains an internal poly(A) region in its 3' end.

In this study, we detected a Japanese isolate of hibiscus latent Fort Pierce virus (HLFPV-J), a member of the genus Tobamovirus, in a hibiscus plant in Japan and determined the complete sequence and organization of its genome. HLFPV-J has four open reading frames (ORFs), each of which shares more than 98 % nucleotide sequence identity with those of other HLFPV isolates. Moreover, HLFPV-J contains a unique internal poly(A) region of variable length, ranging from 44 to 78 nucleotides, in its 3'-untranslated region (UTR), as is the case with hibiscus latent Singapore virus (HLSV), another hibiscus-infecting tobamovirus. The length of the HLFPV-J genome was 6431 nucleotides, including the shortest internal poly(A) region. The sequence identities of ORFs 1, 2, 3 and 4 of HLFPV-J to other tobamoviruses were 46.6-68.7, 49.9-70.8, 31.0-70.8 and 39.4-70.1 %, respectively, at the nucleotide level and 39.8-75.0, 43.6-77.8, 19.2-70.4 and 31.2-74.2 %, respectively, at the amino acid level. The 5'- and 3'-UTRs of HLFPV-J showed 24.3-58.6 and 13.0-79.8 % identity, respectively, to other tobamoviruses. In particular, when compared to other tobamoviruses, each ORF and UTR of HLFPV-J showed the highest sequence identity to those of HLSV. Phylogenetic analysis showed that HLFPV-J, other HLFPV isolates and HLSV constitute a malvaceous-plant-infecting tobamovirus cluster. These results indicate that the genomic structure of HLFPV-J has unique features similar to those of HLSV. To our knowledge, this is the first report of the complete genome sequence of HLFPV.

Presence of poly(A) and poly(A)-rich tails in a positive-strand RNA virus known to lack 3׳ poly(A) tails.

Here we show that Tobacco mosaic virus (TMV), a positive-strand RNA virus known to end with 3׳ tRNA-like structures, does possess a small fraction of gRNA bearing polyadenylate tails. Particularly, many tails are at sites corresponding to the 3׳ end of near full length gRNA, and are composed of poly(A)-rich sequences containing the other nucleotides in addition to adenosine, resembling the degradation-stimulating poly(A) tails observed in all biological kingdoms. Further investigations demonstrate that these polyadenylated RNA species are not enriched in chloroplasts. Silencing of cpPNPase, a chloroplast-localized polynucleotide polymerase known to not only polymerize the poly(A)-rich tails but act as a 3׳ to 5׳ exoribonuclease, does not change the profile of polyadenylate tails associated with TMV RNA. Nevertheless, because similar tails were also detected in other phylogenetically distinct positive-strand RNA viruses lacking poly(A) tails, such kind of polyadenylation may reflect a common but as-yet-unknown interface between hosts and viruses.

Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge.

Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low potency, requiring high dose or boosting to generate a sustained immune response. We have improved the immunogenicity of a plant-made HA vaccine by chemical conjugation to the surface of the Tobacco mosaic virus (TMV) which is non infectious in mammals. We have previously shown that TMV is taken up by mammalian dendritic cells and is a highly effective antigen carrier. In this work, we tested several TMV-HA conjugation chemistries, and compared immunogenicity in mice as measured by anti-HA IgG titers and hemagglutination inhibition (HAI). Importantly, pre-existing immunity to TMV did not reduce initial or boosted titers. Further optimization included dosing with and without alum or oil-in water adjuvants. Surprisingly, we were able to stimulate potent immunogenicity and HAI titers with a single 15 µg dose of HA as a TMV conjugate. We then evaluated the efficacy of the TMV-HA vaccine in a lethal virus challenge in mice. Our results show that a single dose of the TMV-HA conjugate vaccine is sufficient to generate 50% survival, or 100% survival with adjuvant, compared with 10% survival after vaccination with a commercially available H1N1 vaccine. TMV-HA is an effective dose-sparing influenza vaccine, using a single-step process to rapidly generate large quantities of highly effective flu vaccine from an otherwise low potency HA subunit protein.

An infectious RNA with a hepta-adenosine stretch responsible for programmed -1 ribosomal frameshift derived from a full-length cDNA clone of Hibiscus latent Singapore virus.

Hibiscus latent Singapore virus (HLSV) is a member of Tobamovirus and its full-length cDNA clones were constructed. The in vitro transcripts from two HLSV full-length cDNA clones, which contain a hepta-adenosine stretch (pHLSV-7A) and an octo-adenosine stretch (pHLSV-8A), are both infectious. The replication level of HLSV-7A in Nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of HLSV-8A. The replicase proteins of HLSV-7A were produced through programmed -1 ribosomal frameshift (-1 PRF) and the 7A stretch was a slippery sequence for -1 PRF. Mutations to the downstream pseudoknot of 7A stretch showed that the pseudoknot was not required for the frameshift in vitro. The stretch was found to be extended to 8A after subsequent replication cycles in vivo. It is envisaged that HLSV employs the monotonous runs of A and -1 PRF to convert its 7A to 8A to reach higher replication for its survival in plants.

Crystallization and preliminary X-ray crystallographic analysis of the inhibitory domain of the tomato mosaic virus resistance protein Tm-1.

Tm-1, an inhibitor protein of Tomato mosaic virus RNA replication, contains two conserved domains: an uncharacterized domain at its N-terminus and a TIM-barrel-like domain at its C-terminus. The N-terminal domain of Tm-1 has an inhibitory activity and its three-dimensional structure has not been determined. Here, the crystallization and preliminary X-ray diffraction of the N-terminal domain of Tm-1 are reported. A three-wavelength MAD data set was collected from a selenomethionine-labelled crystal and processed to 2.7 Å resolution. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 77.97, b = 105.28, c = 110.62 Å, α = 94.6, ß = 109.3, γ = 108.0°.

The tobamovirus Turnip Vein Clearing Virus 30-kilodalton movement protein localizes to novel nuclear filaments to enhance virus infection.

Plant viruses overcome the barrier of the plant cell wall by encoding cell-to-cell movement proteins (MPs), which direct newly replicated viral genomes to, and across, the wall. The paradigm for how a single MP regulates and coordinates these activities is the Tobacco mosaic virus (TMV) 30-kDa protein (MP(TMV)). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MP(TMV) with endoplasmic reticulum (ER) membrane, microtubules, and plasmodesmata throughout the course of infection. As TMV poorly infects Arabidopsis thaliana, Turnip vein clearing virus (TVCV) is the tobamovirus of choice for studies in this model plant. A key problem, which has contributed to confusion in the field, is the unproven assumption that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed a functional fluorescence-tagged MP(TVCV) and report here the unexpected discovery that MP(TVCV), beyond localizing to ER membrane and plasmodesmata, targeted to the nucleus in a nuclear localization signal (NLS)-dependent manner, where it localized to novel F-actin-containing filaments that associated with chromatin. The MP(TVCV) NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MP(TVCV) was necessary for efficient TVCV cell-to-cell movement and systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. Our studies identify a novel nuclear stage in TVCV infection and suggest that nuclear MP encoded by TVCV and other subgroup 3 tobamoviruses interacts with F-actin and chromatin to modulate host defenses or cellular physiology to favor virus movement and infection.

Guanylylation-competent replication proteins of Tomato mosaic virus are disulfide-linked.

The 130-kDa and 180-kDa replication proteins of Tomato mosaic virus (ToMV) covalently bind guanylate and transfer it to the 5' end of RNA to form a cap. We found that guanylylation-competent ToMV replication proteins are in membrane-bound, disulfide-linked complexes. Guanylylation-competent replication proteins of Brome mosaic virus and Cucumber mosaic virus behaved similarly. To investigate the roles of disulfide bonding in the functioning of ToMV replication proteins, each of the 19 cysteine residues in the 130-kDa protein was replaced by a serine residue. Interestingly, three mutant proteins (C179S, C186S and C581S) failed not only to be guanylylated, but also to bind to the replication template and membranes. These mutants could trans-complement viral RNA replication. Considering that ToMV replication proteins recognize the replication templates, bind membranes, and are guanylylated in the cytoplasm that provides a reducing condition, we discuss the roles of cysteine residues and disulfide bonds in ToMV RNA replication.

Structure of hibiscus latent singapore virus by fiber diffraction: a nonconserved his122 contributes to coat protein stability.

Hibiscus latent Singapore virus (HLSV) is a rigid rod-shaped plant virus and a new member of the Tobamovirus family. Unlike all other Tobamoviruses, the HLSV genome contains a unique poly(A) tract in its 3' untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis. We have determined the structure of HLSV at 3.5 Å by X-ray fiber diffraction and refined it to an R-factor of 0.096. While the overall structure of the HLSV CP resembles that of other Tobamoviruses, there are a few unique differences. There is a kink in the LR helix due to the presence of His122. Also, the adjacent Lys123 may further destabilize the helix by positive charge repulsion, making the kink more pronounced. The His122-Asp88 salt bridge provides significant stability to the loop adjacent to the RR helix. Carboxyl-carboxylate interactions that drive viral disassembly are also different in HLSV. The nucleotide recognition mechanisms for virus assembly between HLSV and ribgrass mosaic virus are similar, but different between tobacco mosaic virus and cucumber green mottle mosaic virus.

Fabrication of aligned magnetic nanoparticles using tobamoviruses.

We used genetically modified tube-shaped tobamoviruses to produce 3 nm aligned magnetic nanoparticles. Amino acid residues facing the central channel of the virus were modified to increase the number of nucleation sites. Energy dispersive X-ray spectroscopy and superconducting quantum interference device analysis suggest that the particles consisted of Co-Pt alloy. The use of tobamovirus mutants is a promising approach to making a variety of components that can be applied to fabricate nanometer-scaled electronic devices.

Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shear flow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (approximately 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.

Effects of brefeldin A on the localization of Tobamovirus movement protein and cell-to-cell movement of the virus.

It has been demonstrated that the subcellular location of Tobamovirus movement protein (MP) which was fused with green fluorescent protein (MP:GFP) changed during the infection process. However, the intracellular route through which MP is transported and its biological meaning are still obscure. Treatment with brefeldin A (BFA), which disrupts ER-to-Golgi transport, inhibited the formation of irregularly shaped and filamentous structures of MP. In this condition, MP was still targeted to plasmodesmata in leaf cells. Furthermore, the viral cell-to-cell movement was not inhibited by BFA treatment. These data indicated that the targeting of viral replication complexes (VRCs) to plasmodesmata is mediated by a BFA-insensitive pathway and that the ER-to-Golgi transport pathway is not involved in viral intercellular movement.

Determination of complete nucleotide sequence of Hibiscus latent Singapore virus: evidence for the presence of an internal poly(A) tract.

We have sequenced the complete genome of a hibiscus-infecting tobamovirus, Hibiscus latent Singapore virus (HLSV). The experimental host range of HLSV is similar to that of another distinct species of hibiscus infecting tobamovirus, Hibiscus latent Fort Pierce virus (HLFPV). The genomic structure of HLSV is similar to other tobamoviruses in general. It consists of a 5' untranslated region (UTR), followed by ORFs encoding for a 128 kDa protein and a 186 kDa readthrough protein, a 30 kDa movement protein (MP), 18 kDa coat protein (CP) and a 3' UTR. The unique feature of HLSV is the presence of a poly(A) tract within its 3' UTR. In our previous work, we have reported MP and CP sequences of HLSV and its phylogenetic analysis. Here we report the complete nucleotide sequence of HLSV, phylogenetic analysis of the nucleotide and amino acid sequences of 128/186 kDa ORFs and the presence of a uniquely located poly(A) tract within the 3' UTR.

Hibiscus virus S is a new subgroup II tobamovirus: evidence from its unique coat protein and movement protein sequences.

The coat protein (CP) and movement protein (MP) sequences of a new tobamovirus infecting Hibiscus rosa-sinensis L were determined. The CP gene encodes 163 amino acid (aa) residues and with a theoretical molecular weight of 18.19 kDa. The MP gene encodes 282 amino acids and its theoretical molecular weight is 30.36 kDa. The nucleotide (nt) and aa sequences of the CP were 46.88 % to 51.63 % and 45.34 % to 57.06 % identical to other tobamoviruses, respectively. The nt and aa sequence identities of MP ranged from 38.81 % to 43.90 % and 30.85 % to 37.88 %, respectively. The predicted virion origin of assembly (OAS) was located in the CP gene. Phylogenetic trees generated based on the nt and aa sequences of both CP and MP genes indicate that this new virus clusters with members of subgroup II of tobamoviruses. Although this hibiscus virus shared a high nt and aa sequence identity with Sunn-hemp mosaic virus (SHMV), Western analysis showed that it is serologically unrelated to SHMV. We propose the name Hibiscus virus S (HVS) for this Singapore isolate. This is the first report on partial nt sequence of a tobamovirus that infects hibiscus.

The tomato mosaic tobamovirus movement protein interacts with a putative transcriptional coactivator KELP.

Viral movement through plasmodesmata in host plants likely depends on the interaction between virus-encoded movement protein (MP) and host proteins. In order to search for MP-interacting protein (MIP), we carried out far-western screening of a Brassica campestris cDNA library using a recombinant MP of tomato mosaic tobamovirus (ToMV) as a probe. One of the positive clones, designated MIP102, was found to be a putative orthologue for a transcriptional coactivator KELP of Arabidopsis thaliana. In vitro analysis with recombinant proteins revealed that ToMV MP could bind to KELP proteins that are derived from different plant species. At least 31 amino acids from the carboxyl-terminus of ToMV MP were dispensable for the interaction with KELP. Other MPs, derived from crucifer tobamovirus CTMV-W and cucumber mosaic cucumovirus, also exhibited comparable binding abilities. This suggests that these MPs could commonly interact with KELP, possibly to modulate the host gene expression.

In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

Rapid simultaneous detection of two orchid viruses using LC- and/or MALDI-mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry are capable of providing molecular mass information on biological samples with high speed, accuracy and sensitivity. With mass spectrometry, identifying a virus based on the molecular weight of its coat protein is relatively simple and accurate. The technique can be applied to all viruses with known coat protein molecular weights. Using the LC/MS and/or MALDI, this paper describes rapid simultaneous detection of the two most prevalent orchid viruses, namely cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV). The coat protein molecular weights of CymMV and ORSV were detected accurately using an extract from 1 g of virus-infected Oncidium orchid flower. Because LC/MS and MALDI allow automated analyses of multiple samples with simple preparation steps, both techniques are ideal for rapid identification of viruses from a large number of samples. This is the first report on the application of LC/MS and/or MALDI for simultaneous detection of two plant viruses from an infected plant extract.

Movement and subcellular localization of a tobamovirus in Arabidopsis.

Tobamoviruses represent a well-characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus-plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis. Here, we describe infection of Arabidopsis by a recently discovered crucifer-infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis, causing mild and fast-developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus-plant interaction.

Similarities between the secondary structure of satellite tobacco mosaic virus and tobamovirus RNAs.

The secondary structure of satellite tobacco mosaic virus (STMV) RNA was predicted using computer simulations of RNA folding. The analogies of structural elements in the 3' end untranslated regions (3'-UTR) of tobamoviral RNAs were analysed. In addition to the tRNA-like structure and pseudoknot stalk, which are found in all known RNAs of tobamoviruses and STMV, another region of stable consecutive pseudoknots was predicted in the 3'-UTR of STMV RNA. A similar pattern of repeated structural units, containing pseudoknot stalks and parts of the tRNA-like structure, was also found in odontoglossum ringspot virus (ORSV) RNA 3'-UTR. The predictions on the structure are supported by sequence comparisons which point to an important functional role of 3' terminal pseudoknots in STMV RNA as well as in other tobamoviral RNAs. The possible participation of pseudoknotted structures in the interactions with coat protein in STMV is discussed.