MPB2C,a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein

The movement protein of tobacco mosaic virus, MP30, mediates viral cell-to-cell transport via plasmodesmata. The complex MP30 intra- and intercellular distribution pattern includes localization to the endoplasmic reticulum, cytoplasmic bodies, microtubules, and plasmodesmata and likely requires interaction with plant endogenous factors. We have identified and analyzed an MP30-interacting protein, MPB2C, from the host plant Nicotiana tabacum. MPB2C constitutes a previously uncharacterized microtubule-associated protein that binds to and colocalizes with MP30 at microtubules. In vivo studies indicate that MPB2C mediates accumulation of MP30 at microtubules and interferes with MP30 cell-to-cell movement. In contrast, intercellular transport of a functionally enhanced MP30 mutant, which does not accumulate and colocalize with MP30 at microtubules, is not impaired by MPB2C. Together, these data support the concept that MPB2C is not required for MP30 cell-to-cell movement but may act as a negative effector of MP30 cell-to-cell transport activity.     

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein

Cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C-terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP-plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP-plasmodesmata interactions in a host-dependent fashion.     

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.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus

We have recently used a green fluorescent protein (GFP) fusion to the gammab protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The gammab protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAbeta but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, betab (TGB1), betac (TGB3), and betad (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.     

Requirements for cell-to-cell movement of Barley stripe mosaic virus in monocot and dicot hosts

The Barley stripe mosaic virus (BSMV) RNAß genome contains a series of overlapping open reading frames termed the triple gene block. The three most abundant proteins, βb, βc and βd, have been shown to have essential roles in infectivity, but their function in cell-to-cell movement has not previously been unambiguously defined, nor has the role of a minor translational read-through protein, βd' been characterized. We have now examined the direct involvement of each of these proteins in cell-to-cell movement in planta by engineering fusions of the green fluorescent protein (GFP) to a cysteine-rich regulatory protein designated γb. Microscopic examination of inoculated and systemically infected barley and oat leaves revealed high levels of fluorescence that moved rapidly through the compact striate vascular tissue without infecting epidermal cells. In contrast, a radial pattern of fluorescence spread through a large number of epidermal and mesophyll cells before entry into the reticulate vascular tissue of the dicot hosts Nicotiania benthamiana and Chenopodium amaranticolor . Mutational analyses indicated that the βb, βc and βd proteins are each essential for cell-to-cell movement in local lesion and systemic hosts, whereas the βd' protein is dispensable. Collectively, these results demonstrate conclusively that the three major triple gene block-encoded proteins act in concert to mediate cell-to-cell movement of BSMV.     

Conversion in the requirement of coat protein in cell-to-cell movement mediated by the cucumber mosaic virus movement protein

Plant viruses have movement protein (MP) gene(s) essential for cell-to-cell movement in hosts. Cucumber mosaic virus (CMV) requires its own coat protein (CP) in addition to the MP for intercellular movement. Our present results using variants of both CMV and a chimeric Brome mosaic virus with the CMV MP gene revealed that CMV MP truncated in its C-terminal 33 amino acids has the ability to mediate viral movement independently of CP. Coexpression of the intact and truncated CMV MPs extremely reduced movement of the chimeric viruses, suggesting that these heterogeneous CMV MPs function antagonistically. Sequential deletion analyses of the CMV MP revealed that the dispensability of CP occurred when the C-terminal deletion ranged between 31 and 36 amino acids and that shorter deletion impaired the ability of the MP to promote viral movement. This is the first report that a region of MP determines the requirement of CP in cell-to-cell movement of a plant virus.     

The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus, a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.     

Susceptibility and symptom development in Arabidopsis thaliana to Tobacco mosaic virus is influenced by virus cell-to-cell movement

To identify host factors that regulate susceptibility to Tobacco mosaic virus (TMV), 14 Arabidopsis thaliana ecotypes were screened for their ability to support TMV systemic movement. The susceptibility phenotypes observed included one ecotype that permitted rapid TMV movement accompanied by symptoms, nine ecotypes that allowed a slower intermediate rate of systemic movement without symptoms, and four ecotypes that allowed little or no systemic TMV movement. Molecular comparisons between ecotypes representing the rapid (Shahdara), intermediate (Col-1), and slow (Tsu-1) movement phenotypes revealed a positive correlation between the ability of TMV to move cell to cell and its speed of systemic movement. Additionally, protoplasts prepared from all three ecotypes supported similar levels of TMV replication, indicating that viral replication did not account for differences in systemic movement. Furthermore, induction of the pathogenesis-related genes PR-1 and PR-5 occurred only in the highly susceptible ecotype Shahdara, demonstrating that reduced local and systemic movement in Col-1 and Tsu-1 was not due to the activation of known host defense responses. Genetic analysis of F2 progeny derived from crosses made between Shahdara and Tsu-1 or Col-1 and Tsu-1 showed the faster cell-to-cell movement phenotypes of Shahdara and Col-1 segregated as single dominant genes. In addition, the Shahdara symptom phenotype segregated independently as a single recessive gene. Taken together, these findings suggest that, within Arabidopsis ecotypes, at least two genes modulate susceptibility to TMV.     

Attenuated strains of tobacco mosaic virus. Reduced synthesis of a viral protein with a cell-to-cell movement function

Attenuated strains of tobacco mosaic virus (TMV) have been used to protect crops against virulent strains. The synthesis of viral proteins and RNAs was investigated in protoplasts that had been infected separately with three tomato strains of TMV, virulent type L, and attenuated strains L11 and L11A. It was revealed that the mutations, which are responsible for the viral attenuation and have been mapped in the p126 (p184) gene, caused a reduction of the synthesis of the viral-coded p30 protein with a cell-to-cell movement function and its mRNA, but it had no significant effect on the synthesis of other viral proteins and RNAs in virus-infected protoplasts. Thus, it was shown that the attenuated strains can multiply as efficiently as the virulent strain in initially inoculated cells, but they can not spread efficiently outside the infected cells. In addition, it is suggested that a non-structural protein, p126 or p184, of TMV is involved in the synthesis of viral subgenomic p30 mRNA.     

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.     

Tomato yellow leaf curl virus V3 protein traffics along microfilaments to plasmodesmata to promote virus cell-to-cell movement

Science China Life Sciences -     

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.     

Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.     

Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication

We have investigated the function of the 30 kd protein of tobacco mosaic virus (TMV) by a reverse genetics approach. First, a point mutation of TMV Ls1 (a temperature-sensitive mutant defective in cell-to-cell movement), that causes an amino acid substitution in the 30 kd protein, was introduced into the parent strain, TMV L. The generated mutant showed the same phenotype as TMV Ls1, and therefore the one-base substitution in the 30 kd protein gene adequately explains the defectiveness of TMV Ls1. Next, four kinds of frame-shift mutants were constructed, whose mutations are located at three different positions of the 30 kd protein gene. All the frame-shift mutants were replication-competent in protoplasts but none showed infectivity on tobacco plants. From these observations the 30 kd protein was confirmed to be involved in cell-to-cell movement. To clarify that the 30 kd protein is not necessary for replication, two kinds of deletion mutants were constructed; one lacking most of the 30 kd protein gene and the other lacking both the 30 kd and coat protein genes. Both mutants replicated in protoplasts and the former still produced the subgenomic mRNA for the coat protein. These results clearly showed that the 30 kd protein, as well as the coat protein, is dispensable for replication and that no cis-acting element for replication is located in their coding sequences. It is also suggested that the signal for coat protein mRNA synthesis may be located within about 100 nucleotides upstream of the initiation codon of the coat protein gene.     

Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.