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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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The effects of Cys-to-Ala substitutions in TGBp3 on virus cell-to-cell movement and TGBp2- and TGBp3-dependent PD targeting of TGBp1.(A) Diagrammatic representation of the infectious plasmid clones of wild-type (WT) and mutant BaMV. Each plasmid clone of BaMV contains a full-length BaMV genome inserted downstream the Cauliflower mosaic virus (CaMV) 35S promoter (as indicated by arrow). pCBG is the WT plasmid clone of BaMV derived from pCB; it has a green fluorescence protein (GFP) gene inserted between the coding sequences of TGBp3 and capsid protein (CP). pGC31A, pGC46A and PGC31, 46A are the three mutant BaMV with either one or both of the conserved cysteine residues (Cys-31 and Cys-46 are indicated by star) in the C-terminal tail of TGBp3 being replaced with alanine. (B) Effects of Cys-to-Ala substitutions in TGBp3 on cell-to-cell movement of BaMV. The fluorescence image of mesophyll cells of C. quinoa leaves inoculated with the WT plasmid clone of BaMV (pCB or pCBG) or either one of the mutant clones of BaMV ( pGC31A, pGC46A, and pGC31, 46A) was taken at 10 dpi. The white bar shown in bottom right corner of each panel is equal to 50 µm. (C) Effects of the Cys-to-Ala substitutions in TGBp3 on TGBp2- and TGBp3-dependent PD targeting of TGBp1. Co-expression of TGBp2 and either the WT or mutant TGBp3:HA with YFP:TGBp1 was conducted by introducing the plasmid (pBA-p2/p3HA, pBA-p2/P3C31AHA, pBA-p2/p3C46AHA or pBA-p2/p3C31, 46AHA) which expresses TGBp2 as well as the WT or mutant TGBp3:HA, and the plasmid (pBA-Y-p1) which expresses YFP:TGBp1 into N. benthamiana by agroinfiltration. The arrows point to the co-localized YFP:TGBp1 and callose in the PD. The white bar in the bottom right corner of each panel indicates the length of 10 µm.
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ppat-1003405-g009: The effects of Cys-to-Ala substitutions in TGBp3 on virus cell-to-cell movement and TGBp2- and TGBp3-dependent PD targeting of TGBp1.(A) Diagrammatic representation of the infectious plasmid clones of wild-type (WT) and mutant BaMV. Each plasmid clone of BaMV contains a full-length BaMV genome inserted downstream the Cauliflower mosaic virus (CaMV) 35S promoter (as indicated by arrow). pCBG is the WT plasmid clone of BaMV derived from pCB; it has a green fluorescence protein (GFP) gene inserted between the coding sequences of TGBp3 and capsid protein (CP). pGC31A, pGC46A and PGC31, 46A are the three mutant BaMV with either one or both of the conserved cysteine residues (Cys-31 and Cys-46 are indicated by star) in the C-terminal tail of TGBp3 being replaced with alanine. (B) Effects of Cys-to-Ala substitutions in TGBp3 on cell-to-cell movement of BaMV. The fluorescence image of mesophyll cells of C. quinoa leaves inoculated with the WT plasmid clone of BaMV (pCB or pCBG) or either one of the mutant clones of BaMV ( pGC31A, pGC46A, and pGC31, 46A) was taken at 10 dpi. The white bar shown in bottom right corner of each panel is equal to 50 µm. (C) Effects of the Cys-to-Ala substitutions in TGBp3 on TGBp2- and TGBp3-dependent PD targeting of TGBp1. Co-expression of TGBp2 and either the WT or mutant TGBp3:HA with YFP:TGBp1 was conducted by introducing the plasmid (pBA-p2/p3HA, pBA-p2/P3C31AHA, pBA-p2/p3C46AHA or pBA-p2/p3C31, 46AHA) which expresses TGBp2 as well as the WT or mutant TGBp3:HA, and the plasmid (pBA-Y-p1) which expresses YFP:TGBp1 into N. benthamiana by agroinfiltration. The arrows point to the co-localized YFP:TGBp1 and callose in the PD. The white bar in the bottom right corner of each panel indicates the length of 10 µm.

Mentions: To have a more direct link of the TGBp3-TGBp2-virion complex to virus movement, the effects of mutations of TGBp2 and TGBp3 on virus movement have to be examined. The Cys-to-Ala substitutions in BaMV TGBp2 have been reported to render the cell-to-cell movement of BaMV relatively inefficient and systemic movement severely inhibited [25]. To see the importance of TGBp3 to virus movement, three mutant BaMV having either one or both of the two conserved cysteine residues (Cys-31 and Cys-46) being replaced with alanine were constructed by polymerase chain reaction (PCR) using the wild-type (WT) plasmid clone of BaMV, pCBG, as template (Figure 9A). Our results showed that all of the three mutant BaMV lost their infectivity (Figure S3) and that the loss of infectivity was neither due to the defect of mutant BaMV in replication (Figure S4A) nor to the defect of TGBp3 expression (Figure S4B). Since the WT and mutant BaMV all contain an expressible green fluorescent protein (GFP) gene in their genome (Figure 9A), green fluorescence spread in mesophyll cells was expected if the tested BaMV has the ability move from cell to cell. As shown in Figure 9B, green fluorescence spread was observed in mesophyll cells of C. quinoa leaves 10 dpi with the WT (pCBG); however, it was restricted in a single cell as the leaves were inoculated with pGC31A, pGC46A or pGC31, 46A. These results clearly demonstrated that the two conserved cysteine residues, Cys-31 and Cys-46, of TGBp3 are essential for cell-to-cell movement of BaMV.


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)

The effects of Cys-to-Ala substitutions in TGBp3 on virus cell-to-cell movement and TGBp2- and TGBp3-dependent PD targeting of TGBp1.(A) Diagrammatic representation of the infectious plasmid clones of wild-type (WT) and mutant BaMV. Each plasmid clone of BaMV contains a full-length BaMV genome inserted downstream the Cauliflower mosaic virus (CaMV) 35S promoter (as indicated by arrow). pCBG is the WT plasmid clone of BaMV derived from pCB; it has a green fluorescence protein (GFP) gene inserted between the coding sequences of TGBp3 and capsid protein (CP). pGC31A, pGC46A and PGC31, 46A are the three mutant BaMV with either one or both of the conserved cysteine residues (Cys-31 and Cys-46 are indicated by star) in the C-terminal tail of TGBp3 being replaced with alanine. (B) Effects of Cys-to-Ala substitutions in TGBp3 on cell-to-cell movement of BaMV. The fluorescence image of mesophyll cells of C. quinoa leaves inoculated with the WT plasmid clone of BaMV (pCB or pCBG) or either one of the mutant clones of BaMV ( pGC31A, pGC46A, and pGC31, 46A) was taken at 10 dpi. The white bar shown in bottom right corner of each panel is equal to 50 µm. (C) Effects of the Cys-to-Ala substitutions in TGBp3 on TGBp2- and TGBp3-dependent PD targeting of TGBp1. Co-expression of TGBp2 and either the WT or mutant TGBp3:HA with YFP:TGBp1 was conducted by introducing the plasmid (pBA-p2/p3HA, pBA-p2/P3C31AHA, pBA-p2/p3C46AHA or pBA-p2/p3C31, 46AHA) which expresses TGBp2 as well as the WT or mutant TGBp3:HA, and the plasmid (pBA-Y-p1) which expresses YFP:TGBp1 into N. benthamiana by agroinfiltration. The arrows point to the co-localized YFP:TGBp1 and callose in the PD. The white bar in the bottom right corner of each panel indicates the length of 10 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675025&req=5

ppat-1003405-g009: The effects of Cys-to-Ala substitutions in TGBp3 on virus cell-to-cell movement and TGBp2- and TGBp3-dependent PD targeting of TGBp1.(A) Diagrammatic representation of the infectious plasmid clones of wild-type (WT) and mutant BaMV. Each plasmid clone of BaMV contains a full-length BaMV genome inserted downstream the Cauliflower mosaic virus (CaMV) 35S promoter (as indicated by arrow). pCBG is the WT plasmid clone of BaMV derived from pCB; it has a green fluorescence protein (GFP) gene inserted between the coding sequences of TGBp3 and capsid protein (CP). pGC31A, pGC46A and PGC31, 46A are the three mutant BaMV with either one or both of the conserved cysteine residues (Cys-31 and Cys-46 are indicated by star) in the C-terminal tail of TGBp3 being replaced with alanine. (B) Effects of Cys-to-Ala substitutions in TGBp3 on cell-to-cell movement of BaMV. The fluorescence image of mesophyll cells of C. quinoa leaves inoculated with the WT plasmid clone of BaMV (pCB or pCBG) or either one of the mutant clones of BaMV ( pGC31A, pGC46A, and pGC31, 46A) was taken at 10 dpi. The white bar shown in bottom right corner of each panel is equal to 50 µm. (C) Effects of the Cys-to-Ala substitutions in TGBp3 on TGBp2- and TGBp3-dependent PD targeting of TGBp1. Co-expression of TGBp2 and either the WT or mutant TGBp3:HA with YFP:TGBp1 was conducted by introducing the plasmid (pBA-p2/p3HA, pBA-p2/P3C31AHA, pBA-p2/p3C46AHA or pBA-p2/p3C31, 46AHA) which expresses TGBp2 as well as the WT or mutant TGBp3:HA, and the plasmid (pBA-Y-p1) which expresses YFP:TGBp1 into N. benthamiana by agroinfiltration. The arrows point to the co-localized YFP:TGBp1 and callose in the PD. The white bar in the bottom right corner of each panel indicates the length of 10 µm.
Mentions: To have a more direct link of the TGBp3-TGBp2-virion complex to virus movement, the effects of mutations of TGBp2 and TGBp3 on virus movement have to be examined. The Cys-to-Ala substitutions in BaMV TGBp2 have been reported to render the cell-to-cell movement of BaMV relatively inefficient and systemic movement severely inhibited [25]. To see the importance of TGBp3 to virus movement, three mutant BaMV having either one or both of the two conserved cysteine residues (Cys-31 and Cys-46) being replaced with alanine were constructed by polymerase chain reaction (PCR) using the wild-type (WT) plasmid clone of BaMV, pCBG, as template (Figure 9A). Our results showed that all of the three mutant BaMV lost their infectivity (Figure S3) and that the loss of infectivity was neither due to the defect of mutant BaMV in replication (Figure S4A) nor to the defect of TGBp3 expression (Figure S4B). Since the WT and mutant BaMV all contain an expressible green fluorescent protein (GFP) gene in their genome (Figure 9A), green fluorescence spread in mesophyll cells was expected if the tested BaMV has the ability move from cell to cell. As shown in Figure 9B, green fluorescence spread was observed in mesophyll cells of C. quinoa leaves 10 dpi with the WT (pCBG); however, it was restricted in a single cell as the leaves were inoculated with pGC31A, pGC46A or pGC31, 46A. These results clearly demonstrated that the two conserved cysteine residues, Cys-31 and Cys-46, of TGBp3 are essential for cell-to-cell movement of BaMV.

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