HSP90 interacts with VP37 to facilitate the cell-to-cell movement of broad bean wilt virus 2
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ABSTRACT The systemic spread of viruses in plants requires successful viral cell-to-cell movement through plasmodesmata (PD). Viral movement proteins (MPs) interact with cellular proteins to modify and utilize host transport routes. Broad bean wilt virus 2 (BBWV2) moves from cell to cell as a virion through the PD gated by VP37, the MP of BBWV2. However, the host proteins that function in the cell-to-cell movement of BBWV2 remain unclear. In this study, we identified cellular heat shock protein 90 (HSP90) as an interacting partner of VP37. The interaction between HSP90 and VP37 was assessed using the yeast two-hybrid assay, co-immunoprecipitation, and bimolecular fluorescence complementation. Tobacco rattle virus-based virus-induced gene silencing analysis revealed that HSP90 silencing significantly inhibited the systemic spread of BBWV2 in Nicotiana benthamiana plants. Furthermore, in planta treatment with geldanamycin (GDA), an inhibitor of the chaperone function of HSP90, demonstrated the necessity of HSP90 in successful cell-to-cell movement and systemic infection of BBWV2. Interestingly, GDA treatment inhibited the HSP90-VP37 interaction at the PD, resulting in the inhibition of VP37-derived tubule formation through the PD. Our results suggest that the HSP90-VP37 interaction regulates VP37-derived tubule formation through the PD, thereby facilitating the cell-to-cell movement of BBWV2. IMPORTANCE This study highlights the regulatory role of heat shock protein 90 (HSP90) in facilitating the cell-to-cell movement of broad bean wilt virus 2 (BBWV2). HSP90 interacted with VP37, the movement protein of BBWV2, specifically at plasmodesmata (PD). This study demonstrated that the HSP90-VP37 interaction is crucial for viral cell-to-cell movement and the formation of VP37-derived tubules, which are essential structures for virus transport through the PD. The ATP-dependent chaperone activity of HSP90 is integral to this interaction, as demonstrated by the inhibition of virus movement upon treatment with geldanamycin, which disrupts the function of HSP90. These findings elucidate the molecular mechanisms underlying the cell-to-cell movement of plant viruses and highlight the role of HSP90 in viral infection. This study suggests that the chaperone activity of HSP90 may function in changing the conformational structure of VP37, thereby facilitating the assembly and function of virus-induced structures required for viral cell-to-cell movement.Keywords:
Plasmodesma
Movement protein
Bimolecular fluorescence complementation
Chaperone (clinical)
Maize and Arabidopsis thaliana class 1 reversibly glycosylated polypeptides (C1RGPs) are plasmodesmata-associated proteins. Previously, over-expression of Arabidopsis C1RGP AtRGP2 in Nicotiana tabacum was shown to reduce intercellular transport of photoassimilate, resulting in stunted, chlorotic plants, and inhibition of local cell-to-cell spread of tobacco mosaic virus (TMV). Here, we used virus induced gene silencing to examine the effects of reduced levels of C1RGPs in Nicotiana benthamiana. Silenced plants show wild-type growth and development. Intercellular transport in silenced plants was probed using fluorescently labeled TMV and its movement protein, P30. P30 shows increased cell-to-cell movement and TMV exhibited accelerated systemic spread compared to control plants. These results support the hypothesis that C1RGPs act to regulate intercellular transport via plasmodesmata.
Plasmodesma
Movement protein
Tobamovirus
Plant cell
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Cell-to-cell movement of a plant virus requires expression of the movement protein (MP). It has not been fully elucidated, however, how the MP functions in primary infected cells. With the use of a microprojectile bombardment-mediated DNA infection system for Tomato mosaic virus (ToMV), we found that the cotransfected ToMV MP gene exerts its effects in the initially infected cells and in their surrounding cells to achieve multicellular spread of movement-defective ToMV. Five other tobamoviral MPs examined also transcomplemented the movement-defective phenotype of ToMV, but the Cucumber mosaic virus 3a MP did not. Together with the cell-to-cell movement of the mutant virus, a fusion between the MP and an enhanced green fluorescent protein variant (EGFP) expressed in trans was distributed multicellularly and localized primarily in plasmodesmata between infected cells. In contrast, in noninfected sites the MP-EGFP fusion accumulated predominantly inside the bombarded cells as irregularly shaped aggregates, and only a minute amount of the fusion was found in plasmodesmata. Thus, the behavior of ToMV MP is greatly modulated in the presence of a replicating virus and it is highly likely that the MP spreads in the infection sites, coordinating with the cell-to-cell movement of the viral genome.
Plasmodesma
Movement protein
Tobamovirus
Cell fusion
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Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.
Plasmodesma
Movement protein
Plant cell
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For systemic infection of a host plant, viruses multiply in the initially infected cell and spread to the neighbouring cells through plasmodesmata (cell-to-cell movement), to eventually reach the vascular system and use the phloem to spread to other plant parts (long-distance movement). To achieve cell-to-cell transport through plasmodesmata, these complex pores in the plant cell wall must be modulated to allow viral passage. Two major types of cell-to-cell transport have been described, movement of the viral genome in a non-encapsidated form, as exemplified by Tobacco mosaic virus (TMV), and "tubule-guided" movement of mature virus particles (virions), exemplified by Cowpea mosaic virus (CPMV). In both mechanisms one or more virally encoded movement proteins (MP) play an essential role in the targeting of infectious entities from the site of replication to the plasmodesmata, as well as in the subsequent modification of and transport through the modified pores. However, it is generally recognised that intercellular movement is a concerted effort of not only viral factors but also host factors, the knowledge of the latter being very scarce at the moment.With CPMV, the MP polymerises within the plasmodesmal pore into a transport tubule, through which mature virions then are delivered into the neighbouring uninfected cell. Identical tubules are also formed in single plant protoplasts that are infected with CPMV or transfected with the MP gene alone, hence, in the absence of cell wall and plasmodesmata.At the onset of the research presented in this thesis, no information about host proteins interacting with the CPMV MP was available. Such interactions were to be expected, for instance during the process of transport (targeting) of the MP from its site of synthesis to the periphery of the infected cell, the polymerisation process at the plasma membrane, and the structural modification of the plasmodesma. Thus, the research described in this thesis focused on the functioning of the CPMV MP with special emphasis on its interactions with virion proteins and host proteins.For initial studies on these interactions the property of the CPMV MP to assemble into tubules on single cell protoplasts was exploited in Chapter 2. Thus it was shown that virus particles residing in the tubule contain a single deviant species of the small coat protein (S CP) that is larger than the two forms of S CP (S-s and S-f) which are consistently found in virus present in the cytoplasm of infected cells. The nature of the deviation is not known, but the exclusive presence of this deviant S CP in virions that are being transported suggests that the S CP is in some way involved in cell-to-cell movement.Identification of host proteins in isolated tubule fractions by electrophoretic analysis was not successful, but a directed search for potential host proteins by Western blot analysis using specific antibodies indicated the presence of pectin methylesterase (PME) in the plasma membrane surrounding the tubule (Chapter 2). This protein has previously been implicated in cell-to-cell movement of other plant viruses, i.e. TMV, Cauliflower mosaic virus and Turnip vein clearing virus . The PME enzyme is involved in cell wall turnover and affects cell wall rigidity by modulating pH and ion balance. Such cell wall dynamics could be a necessity for the modification of the plasmodesmal pore to enable the insertion of a viral transport tubule.The interaction between the MP and virion proteins was further investigated in Chapter 3. Protein overlay assays and ELISA showed that the MP binds only to its homologous virions and that it is the large (L) coat protein which is involved in this binding. Considering also the deviation found in the S CP of virions within the transport tubules, it is conceivable that both CPs play a crucial but different role in the cell-to-cell movement of CPMV. A C-terminal deletion in MP, which in planta results in a mutant virus defective in cell-to-cell movement and producing tubules devoid of particles, also resulted in the abolishment of L CP binding, thus validating the in vitro binding approaches.The ability of the CPMV MP to bind nucleic acid and rNTP was analysed in Chapter 4. It is shown that MP binds rGTP but no other rNTPs, and by site-directed mutagenesis the GTP binding site was located within a sequence motif conserved among the MPs of tobamo- and comoviruses. The non-GTP-binding mutant MP exhibited disturbed intracellular targeting and tubule formation, suggesting that GTP binding may play a significant role in targeted transport and multimerization of the MP. It was also shown that the MP is capable of binding both ss-RNA and DNA, but not ds nucleic acids.The studies on possible interactions between CPMV MP and host (plasma membrane) proteins were extended in Chapter 5. To identify potential MP-binding host proteins, purified MP was used as a probe in overlay assays and affinity column chromatography to assess plasma membrane proteins for their affinity to the MP. In the blot overlay assays, candidate MP-binding proteins with apparent sizes of 34, 30 and 28 kDa were detected. Further analysis of the cowpea plasma membrane fraction using affinity chromatography also revealed a limited number of eight MP-binding proteins including again a 30 kDa protein band. Sequencing of the 30 kDa protein band revealed that it actually represented a mixture of two protein species, i.e. an aquaporin and a vacuolar-type ATPase. A possible role of these host proteins in viral MP functioning is discussed in Chapter 5.Finally, in the General Discussion (Chapter 6) the results obtained in this thesis research are discussed and integrated in a speculative model for the functioning of the CPMV MP, accommodating the different observed interactions with virion and host proteins.
Plasmodesma
Movement protein
Cowpea mosaic virus
Plant cell
Brome mosaic virus
Mosaic virus
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The interaction between tobacco mosaic virus and its host plant cells has been intensively studied as a model for macromolecular trafficking. The observation that GFP-labelled TMV movement protein localises to microtubules led to the suggestion that microtubules are required for the cell to cell movement of the virus. In a recent paper we have demonstrated that the targeting of TMV movement protein to plasmodesmata requires the actin and ER networks, which supports previous evidence from our laboratory that showed that disruption of microtubules did not prevent cell to cell movement of TMV virus, and that a mutated movement protein, which did not localise to micro-tubules, showed enhanced viral movement. In this addendum we speculate where the TMV movement protein accumulates within plasmodesmata, and the relationship of this accumulation to the cell to cell movement of the virus.
Plasmodesma
Movement protein
Tobamovirus
Plant cell
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Transgenic tobacco plants expressing a gene encoding the tobacco mosaic virus (TMV) movement protein (30K) were studied using immunocytochemical techniques. The movement protein was shown to be localized within or on most of the plasmodesmata observed in the transformed plant. These results are consistent with the idea that the movement protein interacts with the plasmodesmata to facilitate the cell-to-cell spread of TMV.
Plasmodesma
Movement protein
Tobamovirus
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Plasmodesma
Movement protein
Transport protein
Tobamovirus
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After the virus has entered a plant, expressed its genes, and replicated, it must move to and through the plasmodesmata of the infected cell to adjacent cells, as well as into and back out of the vascular tissue during transportation to distal parts of the plant. Each of these steps requires some function associated with viral-encoded proteins. This chapter reviews what is known about intracellular movement of the cucumber mosaic virus (CMV) RNAs and associated proteins, as well as the intercellular movement through plasmodesmata to adjacent cells and systemic movement into the vascular system of the plant, followed by subsequent cellular movement. In addition, CMV proteins and specific sequences identified as being involved in various aspects of virus movement are described, along with the effects of various natural and human-made mutations on these processes and on the subcellular localization of the CMV 3a movement protein mutants.
Plasmodesma
Movement protein
Plant cell
Cucumovirus
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Reversibly glycosylated polypeptides (RGPs) have been identified in many plant species and play an important role in cell wall formation, intercellular transport regulation, and plant–virus interactions. Most plants have several RGP genes with different expression patterns depending on the organ and developmental stage. Here, we report on four members of the RGP family in N. benthamiana. Based on a homology search, NbRGP1-3 and NbRGP5 were assigned to the class 1 and class 2 RGPs, respectively. We demonstrated that NbRGP1–3 and 5 mRNA accumulation increases significantly in response to tobacco mosaic virus (TMV) infection. Moreover, all identified class 1 NbRGPs (as distinct from NbRGP5) suppress TMV intercellular transport and replication in N. benthamiana. Elevated expression of NbRGP1–2 led to the stimulation of callose deposition at plasmodesmata, indicating that RGP-mediated TMV local spread could be affected via a callose-dependent mechanism. It was also demonstrated that NbRGP1 interacts with TMV movement protein (MP) in vitro and in vivo. Therefore, class 1 NbRGP1–2 play an antiviral role by impeding intercellular transport of the virus by affecting plasmodesmata callose and directly interacting with TMV MP, resulting in the reduced viral spread and replication.
Plasmodesma
Callose
Tobamovirus
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Movement protein
Plasmodesma
Potato virus X
Tobamovirus
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Citations (78)