Interaction of Sesbania mosaic virus movement protein with the coat protein--implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N-terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.     

Interaction of tomato mosaic virus movement protein with tobacco RIO kinase

Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plasmodesmata. To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method. One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel protein kinase, Rio1p. We isolated a full-length cDNA by RT-PCR. The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively. In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP derived from Cucumber mosaic virus. NtRIO had autophosphorylation activity and phosphorylated ToMV MP. Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO. The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO.     

Radish mosaic virus VPg Characteristics and linkage with virion RNAs

Comoviruses have a bipartite RNA genome. It is suggested that a small protein, VPg, is covalently linked to both RNAs. We have found that both radish mosaic virus RNAs are linked to identical VPg molecules via a phosphodiester bond between their 5′-terminal nucleotides and a serine residue of VPg.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.     

Dimerization of recombinant tobacco mosaic virus movement protein

The p30 movement protein (MP) is essential for cell-to-cell spread of tobacco mosaic virus in planta. We used anion-exchange chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain highly purified 30-kDa MP, which migrated as a single band in native PAGE. Analytical ultracentrifugation suggested that the protein was monodisperse and dimeric in the nonionic detergent n-octyl-beta-D-glucopyranoside. Circular dichroism (CD) spectroscopy showed that the detergent-solubilized protein contained significant alpha-helical secondary structure. Proteolysis of the C-tail generated a trypsin-resistant core that was a mixture of primarily monomers and some dimers. We propose that MP dimers are stabilized by electrostatic interactions in the C terminus as well as hydrophobic interactions between putative transmembrane alpha-helical coiled coils.     

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.     

The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein

The P30 protein of tobacco mosaic virus (TMV) is required for cell to cell movement of viral RNA, which presumably occurs through plant intercellular connections, the plasmodesmata. The mechanism by which P30 mediates transfer of TMV RNA molecules through plasmodesmata channels is unknown. We have identified P30 as an RNA and single-stranded (ss) DNA binding protein. Binding of purified P30 to ss nucleic acids is strong, highly cooperative, and sequence nonspecific with a minimal binding site of 4-7 nucleotides per P30 monomer. In-frame deletions across P30 were used to localize the ss nucleic acid binding domain to within amino acid residues 65-86 of the protein. We propose that binding of P30 to TMV RNA creates an unfolded protein-RNA complex that functions as an intermediate in virus cell to cell movement through plasmodesmata.     

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.     

Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F

The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (K(a)) and average binding sites (n) for VPg were (8.51 +/- 0.21) x 10(6) M(-1) and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative DeltaH degrees (-29.32 +/- 0.13 kJmol(-1)) and positive DeltaS degrees (36.88 +/- 0.25 Jmol(-1)K(-1)). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m(7)G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 +/- 1.27 and 75.37 +/- 2.95 kJmol(-1) respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells

In a functional genomic screen performed by combining an Arabidopsis–yellow fluorescent protein (YFP)-fused complementary DNA (cDNA) library, rat fibroblasts as host and automatic microscopy, we found a short protein with a predictable trans-membrane domain encoded on chromosome 2. In rat fibroblasts, its pattern of distribution was to various organelle-like structures. From the databases, we learned that it has another family member in Arabidopsis and homologs in several other plants, Chlamydomonas and fungi, with a highly conserved N-terminal region. We named this protein from Arabidopsis short membrane protein (SMP) 2. No SMP homologs were found in mammalian sequence databases. When the full-length cDNAs of SMP2 was fused to YFP under the 35S promoter, comparable distribution was observed in Nicotiana benthamiana leaves, suggesting an unknown, evolutionarily conserved localization signal. Similar localization was observed when SMP2 was expressed in N. benthamiana leaves under the control of its own 5′ regulatory sequences. Colocalization studies with green fluorescent protein and red fluorescent protein chimeras revealed its colocalization with chloroplasts, peroxisomes, and mitochondria. No localization of SMP2 was observed in the Golgi. Immunostaining with specific antibodies corroborated the SMP2 localization to the three organelles.     

Cowpea mosaic virus VPg: sequencing of radiochemically modified protein allows mapping of the gene on B RNA

A partial amino acid sequence of cowpea mosaic virus (CPMV) VPg radiochemically modified by chloramine-T and Bolton-Hunter reagent has been determined. VPg covalently bound to viral RNA chains (VPg-RNA) was iodinated with chloramine-T and Bolton-Hunter reagent to label tyrosine and lysine residues, respectively. [¹²⁵I]VPg-RNA was digested with nuclease P1 and the resulting [¹²⁵I]VPg-pU was purified by SDS-polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Control experiments with chemically synthesized poliovirus VPg showed the feasibility of radiochemical microsequence analysis of protein that had been radiochemically modified by chloramine-T and Bolton-Hunter reagent. Analysis of CPMV [¹²⁵I]VPg-pU revealed the presence of tyrosine residues at position 12 and 14, and of lysine residues at position 3 and 20, respectively. In combination with Edman degradation of unlabeled CPMV VPg, which showed serine and arginine residues to be present at position 1 and 2, respectively, the data obtained allow the precise positioning of VPg within the 200 000 dalton (200 K) polyprotein encoded by CPMV B RNA and the prediction of its entire amino acid sequence. VPg is located at the COOH terminus of its 60 K, membrane-bound,precursor and proximal to the amino terminus of the protease-polymerase domain of the polyprotein. A processing scheme for the 200 K polyprotein is discussed in which Gln-Ser amino acid pairs act as the major signal for proteolytic cleavage.     

Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1

The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.     

Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids.

Cell-to-cell spread of tobacco mosaic virus (TMV) is presumed to occur through plant intercellular connections, the plasmodesmata. Viral movement is an active process mediated by a specific virus-encoded P30 protein. P30 has at least two functions, to cooperatively bind single-stranded nucleic acids and to increase plasmodesmatal permeability. Here, we visualized P30 complexes with single-stranded DNA and RNA. These complexes are long, unfolded, and very thin (1.5 to 2.0 nm in diameter). Unlike TMV virions (300 x 18 nm), the complexes are compatible in size with the P30-induced increase in plasmodesmatal permeability (2.4 to 3.1 nm), making them likely candidates for the structures involved in the cell-to-cell movement of TMV. Mutational analysis using single and double deletion mutants of P30 revealed three regions potentially important for the protein function. Amino acid residues 65 to 86 possibly are required for correct folding of the active protein, and the regions between amino acid residues 112 to 185 and 185 to 268 potentially contain two independently active single-stranded nucleic acid binding domains designated binding domains A and B, respectively.     

Tobacco mosaic virus movement protein enhances the spread of RNA silencing

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.