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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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Co-fractionation of TGBp3, TGBp2 and CP along the Sephacryl S-1000 gel filtration profile.(A) The gel filtration profile of the S100 fractionated by the Sephacryl S-1000 column. A, B, C and D are the four major peaks detected in the profile; 1 to 34 are fraction numbers. (B–E) Immunological analyses of TGBp3:HA, TGBp2, TGBp1 and CP, respectively, in the samples fractionated by the Sephacryl S-1000 column. In B and C, protein bands were detected using a chemiilumination system. In D and E, protein bands were detected using 1-chloro-4-naphathol as a color development agent.
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ppat-1003405-g005: Co-fractionation of TGBp3, TGBp2 and CP along the Sephacryl S-1000 gel filtration profile.(A) The gel filtration profile of the S100 fractionated by the Sephacryl S-1000 column. A, B, C and D are the four major peaks detected in the profile; 1 to 34 are fraction numbers. (B–E) Immunological analyses of TGBp3:HA, TGBp2, TGBp1 and CP, respectively, in the samples fractionated by the Sephacryl S-1000 column. In B and C, protein bands were detected using a chemiilumination system. In D and E, protein bands were detected using 1-chloro-4-naphathol as a color development agent.

Mentions: To further analyze the TGBp3-based complex, the Sarkosyl extracted fraction (S100) was applied onto a Sephacryl S-1000 gel filtration column suitable for separation of large complexes with molecular masses up to 108 (100 MDa) or for separation of spherical particles up to 400 nm. The distributions of TGBp1, TGBp2, TGBp3 and CP along the filtration profile were analyzed by western blot (Figure 5). Apparently, substantial co-fractionation of TGBp3, TGBp1 and CP was observed in the largest peak, peak C. However, substantial co-fractionation of the main protein components required for intracellular virus transport, TGBp2 and TGBp3, and the early eluted CP (all peaked apparently at F11), but not TGBp1, was mainly observed in peak B. These results suggested to us that the TGBp2- and TGBp3-based complex is able to stably associate with the CP-coated virions but not non-virion vRNP which is assumed to possess TGBp1 [29]. To corroborate this idea, three sample fractions (F11, F12 and F14) from peak B and four (F7, F21, F22 and F26) from peaks A, C and D, respectively, were subjected to immunoprecipitation with anti-HA. The immunoprecipitate was then analyzed with anti-TGBp1, anti-TGBp2, anti-HA and anti-CP (Figure 6). As shown in Figure 6A, TGBp3:HA was able to be immunoprecipitated with anti-HA in all the tested fractions, except F26 (peak D). Significant co-immunoprecipitation of TGBp2 (Figure 6B) with TGBp3:HA was mainly significantly detected in fractions (F11, F12 and F14) of peak B. Significant co-immunoprecipitation of CP (Figure 6D) was observed in fractions (F11, F12, F14, F21 and F22) of peaks B and C. However, TGBp1 was unable to be co-immunoprecipitated with TGBp3:HA in all of the tested fractions (Figure 6C). Taken together, these results suggested that peak B contains TGBp2-TGBp3-based complex and BaMV virions rather than non-virion vRNP. In other words, the TGBp2-TGBp3-based complex may be able to associate with CP on the virions. To confirm this idea, the immunoprecipitate was examined with transmission electron microscopy (TEM). Just as expected, virus particles similar to the purified BaMV-S (Figure 6E, left panel) were observed in the immunoprecipitate (Figure 6E, right panel), indicating that the TGBp2-TGBp3-based complex is able to stably associate with virions.


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)

Co-fractionation of TGBp3, TGBp2 and CP along the Sephacryl S-1000 gel filtration profile.(A) The gel filtration profile of the S100 fractionated by the Sephacryl S-1000 column. A, B, C and D are the four major peaks detected in the profile; 1 to 34 are fraction numbers. (B–E) Immunological analyses of TGBp3:HA, TGBp2, TGBp1 and CP, respectively, in the samples fractionated by the Sephacryl S-1000 column. In B and C, protein bands were detected using a chemiilumination system. In D and E, protein bands were detected using 1-chloro-4-naphathol as a color development agent.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003405-g005: Co-fractionation of TGBp3, TGBp2 and CP along the Sephacryl S-1000 gel filtration profile.(A) The gel filtration profile of the S100 fractionated by the Sephacryl S-1000 column. A, B, C and D are the four major peaks detected in the profile; 1 to 34 are fraction numbers. (B–E) Immunological analyses of TGBp3:HA, TGBp2, TGBp1 and CP, respectively, in the samples fractionated by the Sephacryl S-1000 column. In B and C, protein bands were detected using a chemiilumination system. In D and E, protein bands were detected using 1-chloro-4-naphathol as a color development agent.
Mentions: To further analyze the TGBp3-based complex, the Sarkosyl extracted fraction (S100) was applied onto a Sephacryl S-1000 gel filtration column suitable for separation of large complexes with molecular masses up to 108 (100 MDa) or for separation of spherical particles up to 400 nm. The distributions of TGBp1, TGBp2, TGBp3 and CP along the filtration profile were analyzed by western blot (Figure 5). Apparently, substantial co-fractionation of TGBp3, TGBp1 and CP was observed in the largest peak, peak C. However, substantial co-fractionation of the main protein components required for intracellular virus transport, TGBp2 and TGBp3, and the early eluted CP (all peaked apparently at F11), but not TGBp1, was mainly observed in peak B. These results suggested to us that the TGBp2- and TGBp3-based complex is able to stably associate with the CP-coated virions but not non-virion vRNP which is assumed to possess TGBp1 [29]. To corroborate this idea, three sample fractions (F11, F12 and F14) from peak B and four (F7, F21, F22 and F26) from peaks A, C and D, respectively, were subjected to immunoprecipitation with anti-HA. The immunoprecipitate was then analyzed with anti-TGBp1, anti-TGBp2, anti-HA and anti-CP (Figure 6). As shown in Figure 6A, TGBp3:HA was able to be immunoprecipitated with anti-HA in all the tested fractions, except F26 (peak D). Significant co-immunoprecipitation of TGBp2 (Figure 6B) with TGBp3:HA was mainly significantly detected in fractions (F11, F12 and F14) of peak B. Significant co-immunoprecipitation of CP (Figure 6D) was observed in fractions (F11, F12, F14, F21 and F22) of peaks B and C. However, TGBp1 was unable to be co-immunoprecipitated with TGBp3:HA in all of the tested fractions (Figure 6C). Taken together, these results suggested that peak B contains TGBp2-TGBp3-based complex and BaMV virions rather than non-virion vRNP. In other words, the TGBp2-TGBp3-based complex may be able to associate with CP on the virions. To confirm this idea, the immunoprecipitate was examined with transmission electron microscopy (TEM). Just as expected, virus particles similar to the purified BaMV-S (Figure 6E, left panel) were observed in the immunoprecipitate (Figure 6E, right panel), indicating that the TGBp2-TGBp3-based complex is able to stably associate with virions.

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