The SNARE protein Syp71 is essential for turnip mosaic virus infection by mediating fusion of virus-induced vesicles with chloroplasts.
ABSTRACT: All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection.
Project description:Chloroplast-bound vesicles are key components in viral replication complexes (VRCs) of potyviruses. The potyviral VRCs are induced by the second 6?kDa protein (6K2) and contain at least viral RNA and nuclear inclusion protein b. To date, no chloroplast protein has been identified to interact with 6K2 and involve in potyvirus replication. In this paper, we showed that the Photosystem II oxygen evolution complex protein of Nicotiana benthamiana (NbPsbO1) was a chloroplast protein interacting with 6K2 of Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) and present in the VRCs. The first 6?kDa protein (6K1) was recruited to VRCs by 6K2 but had no interaction with NbPSbO1. Knockdown of NbPsbO1 gene expression in N. benthamiana plants through virus-induced gene silencing significantly decreased the accumulation levels of TVBMV and another potyvirus Potato virus Y, but not Potato virus X of genus Potexvirus. Amino acid substitutions in 6K2 that disrupted its interaction with NbPsbO1 also affected the replication of TVBMV. NbPsbP1 and NbPsbQ1, two other components of the Photosystem II oxygen evolution complex had no interaction with 6K2 and no effect on TVBMV replication. To conclude, 6K2 recruits 6K1 to VRCs and hijacks chloroplast protein NbPsbO1 to regulate potyvirus replication.
Project description:To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs). However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV) induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked toward the plasma membrane and were associated with plasmodesmata (PD). We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP) to visualize how 6K2 moved intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.
Project description:The replication of positive-strand RNA viruses occurs in cytoplasmic membrane-bound virus replication complexes (VRCs). Depending on the virus, distinct cellular organelles such as the endoplasmic reticulum (ER), chloroplast, mitochondrion, endosome, and peroxisome are recruited for the formation of VRC-associated membranous structures. Previously, the 6,000-molecular-weight protein (6K) of plant potyviruses was shown to be an integral membrane protein that induces the formation of 6K-containing membranous vesicles at endoplasmic reticulum (ER) exit sites for potyvirus genome replication. Here, we present evidence that the 6K-induced vesicles predominantly target chloroplasts, where they amalgamate and induce chloroplast membrane invaginations. The vesicular transport pathway and actomyosin motility system are involved in the trafficking of the 6K vesicles from the ER to chloroplasts. Viral RNA, double-stranded RNA, and viral replicase components are concentrated at the 6K vesicles that associate with chloroplasts in infected cells, suggesting that these chloroplast-bound 6K vesicles are the site for potyvirus replication. Taken together, these results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.
Project description:Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes, including responses to pathogen attack. Here, we report the identification of Glycine max (soybean) endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a bottom-up proteomics approach. Knockdown of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B in Arabidopsis thaliana (AtDRP2A, AtDRP2B), homologs of GmSDL5A, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both A. thaliana drp2a (atdrp2a) and atdrp2b knockout mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI, and 6K2. Of these viral proteins, VPg, CP, and CI are essential for viral intercellular movement, and 6K2, VPg, and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together, these data suggest that dynamin-like proteins are novel host factors of potyviruses and that endocytic processes are involved in potyvirus infection.IMPORTANCE It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.
Project description:The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.
Project description:P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process.IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses.
Project description:<h4>Unlabelled</h4>Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.<h4>Importance</h4>Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.
Project description:Turnip mosaic virus (TuMV), a potyvirus, is a flexible filamentous plant virus that displays a helical arrangement of coat protein copies (CPs) bound to the ssRNA genome. TuMV is a bona fide representative of the Potyvirus genus, one of most abundant groups of plant viruses, which displays a very wide host range. We have studied by cryoEM the structure of TuMV virions and its viral-like particles (VLPs) to explore the role of the interactions between proteins and RNA in the assembly of the virions. The results show that the CP-RNA interaction is needed for the correct orientation of the CP N-terminal arm, a region that plays as a molecular staple between CP subunits in the fully assembled virion.
Project description:The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.
Project description:The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.