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The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.

Chou YL, Hung YJ, Tseng YH, Hsu HT, Yang JY, Wung CH, Lin NS, Meng M, Hsu YH, Chang BY - PLoS Pathog. (2013)

Bottom Line: Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion.This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions.Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China.

ABSTRACT
The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

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Requirement of TGBp2 and TGBp3 for efficient PD-targeting of TGBp1.(A) PD-targeting of TGBp1 in the presence of TGBp2. mCherry:TGBp1 and YFP:TGBp2 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. The PD of cell periphery was visualized by aniline blue fluorochrome staining. In the most right-hand upper panel, the arrows indicate YFP:TGBp2 co-localized with PD. (B) PD-targeting of TGBp1 in the presence of TGBp3. mCherry:TGBp1 and YFP:TGBp3 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. In the most right-hand upper panel, the arrows indicate the YFP:TGBp3 co-localized with PD. (C) PD-targeting of TGBp1 in the presence of both TGBp2 and TGBp3. Upper panels, the YFP:TGBp1 expressed alone in leaf epidermal cells. Lower panels, the YFP:TGBp1, non-fused TGBp2 and TGBp3 co-expressed in leaf epidermal cells. All confocal images were taken from a single optical plane. Bars, 10 µm.
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ppat-1003405-g008: Requirement of TGBp2 and TGBp3 for efficient PD-targeting of TGBp1.(A) PD-targeting of TGBp1 in the presence of TGBp2. mCherry:TGBp1 and YFP:TGBp2 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. The PD of cell periphery was visualized by aniline blue fluorochrome staining. In the most right-hand upper panel, the arrows indicate YFP:TGBp2 co-localized with PD. (B) PD-targeting of TGBp1 in the presence of TGBp3. mCherry:TGBp1 and YFP:TGBp3 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. In the most right-hand upper panel, the arrows indicate the YFP:TGBp3 co-localized with PD. (C) PD-targeting of TGBp1 in the presence of both TGBp2 and TGBp3. Upper panels, the YFP:TGBp1 expressed alone in leaf epidermal cells. Lower panels, the YFP:TGBp1, non-fused TGBp2 and TGBp3 co-expressed in leaf epidermal cells. All confocal images were taken from a single optical plane. Bars, 10 µm.

Mentions: To realize the biological significance of TGBp1 association with the stable complex of TGBp2-TGBp3-virion, we analyzed whether targeting of the potex-like TGBp1 to PD requires assistance from TGBp3 or from both TGBp2 and TGBp3 as reported for hordei-like viruses [42]–[44]. To this end, agro-compatible plasmid, expressing TGBp1, TGBp2 or TGBp3:HA with a fluorescence-protein (FP) fusion at the N-terminus of each protein, was constructed and used to analyze the TGBp2- and/or TGBp3-dependence of PD-localization of TGBp1 in leaf epidermal cells accompanied with the use of a callose-specific staining dye, aniline blue fluorochrome. The PD-localization of TGBp1 under the co-expression of either TGBp2 or TGBp3 is shown in Figures 8A and 8B. A portion of TGBp2 was localized at the PD (Figure 8A, upper panels). Similar phenomenon was observed for TGBp3; it was either localized at the PD or distributed over the cell periphery (Figure 8B, upper panels). However, TGBp1 was unable to be targeted to the PD while only TGBp2 or TGBp3 was co-expressed. The PD-localization of TGBp1 under the co-expression of both TGBp2 and TGBp3 is shown in Figure 8C. Clearly, TGBp1 was redistributed from cytosol (upper panels) to the PD (lower panels). Thus, both TGBp2 and TGBp3 are essential for efficient targeting of TGBp1 to the PD in potexvirus and the C-terminal HA-tag on TGBp3 does not interfere with TGBp1 recruitment.


The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.

Chou YL, Hung YJ, Tseng YH, Hsu HT, Yang JY, Wung CH, Lin NS, Meng M, Hsu YH, Chang BY - PLoS Pathog. (2013)

Requirement of TGBp2 and TGBp3 for efficient PD-targeting of TGBp1.(A) PD-targeting of TGBp1 in the presence of TGBp2. mCherry:TGBp1 and YFP:TGBp2 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. The PD of cell periphery was visualized by aniline blue fluorochrome staining. In the most right-hand upper panel, the arrows indicate YFP:TGBp2 co-localized with PD. (B) PD-targeting of TGBp1 in the presence of TGBp3. mCherry:TGBp1 and YFP:TGBp3 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. In the most right-hand upper panel, the arrows indicate the YFP:TGBp3 co-localized with PD. (C) PD-targeting of TGBp1 in the presence of both TGBp2 and TGBp3. Upper panels, the YFP:TGBp1 expressed alone in leaf epidermal cells. Lower panels, the YFP:TGBp1, non-fused TGBp2 and TGBp3 co-expressed in leaf epidermal cells. All confocal images were taken from a single optical plane. Bars, 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675025&req=5

ppat-1003405-g008: Requirement of TGBp2 and TGBp3 for efficient PD-targeting of TGBp1.(A) PD-targeting of TGBp1 in the presence of TGBp2. mCherry:TGBp1 and YFP:TGBp2 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. The PD of cell periphery was visualized by aniline blue fluorochrome staining. In the most right-hand upper panel, the arrows indicate YFP:TGBp2 co-localized with PD. (B) PD-targeting of TGBp1 in the presence of TGBp3. mCherry:TGBp1 and YFP:TGBp3 were co-expressed in leaf epidermal cells of N. benthamiana through agro-infiltration. In the most right-hand upper panel, the arrows indicate the YFP:TGBp3 co-localized with PD. (C) PD-targeting of TGBp1 in the presence of both TGBp2 and TGBp3. Upper panels, the YFP:TGBp1 expressed alone in leaf epidermal cells. Lower panels, the YFP:TGBp1, non-fused TGBp2 and TGBp3 co-expressed in leaf epidermal cells. All confocal images were taken from a single optical plane. Bars, 10 µm.
Mentions: To realize the biological significance of TGBp1 association with the stable complex of TGBp2-TGBp3-virion, we analyzed whether targeting of the potex-like TGBp1 to PD requires assistance from TGBp3 or from both TGBp2 and TGBp3 as reported for hordei-like viruses [42]–[44]. To this end, agro-compatible plasmid, expressing TGBp1, TGBp2 or TGBp3:HA with a fluorescence-protein (FP) fusion at the N-terminus of each protein, was constructed and used to analyze the TGBp2- and/or TGBp3-dependence of PD-localization of TGBp1 in leaf epidermal cells accompanied with the use of a callose-specific staining dye, aniline blue fluorochrome. The PD-localization of TGBp1 under the co-expression of either TGBp2 or TGBp3 is shown in Figures 8A and 8B. A portion of TGBp2 was localized at the PD (Figure 8A, upper panels). Similar phenomenon was observed for TGBp3; it was either localized at the PD or distributed over the cell periphery (Figure 8B, upper panels). However, TGBp1 was unable to be targeted to the PD while only TGBp2 or TGBp3 was co-expressed. The PD-localization of TGBp1 under the co-expression of both TGBp2 and TGBp3 is shown in Figure 8C. Clearly, TGBp1 was redistributed from cytosol (upper panels) to the PD (lower panels). Thus, both TGBp2 and TGBp3 are essential for efficient targeting of TGBp1 to the PD in potexvirus and the C-terminal HA-tag on TGBp3 does not interfere with TGBp1 recruitment.

Bottom Line: Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion.This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions.Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China.

ABSTRACT
The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

Show MeSH
Related in: MedlinePlus