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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-immunoprecipitation of various virus components with TGBp3:HA.(A) Immunoprecipitation of the TGBp3:HA-containing membrane protein complex. Inp, the S100 prepared from mock-inoculated (H) and BaMV-infected tissues (I) pre-cleaned with Protein A-Sepharose (PA). IpP and IpS, the immunoprecipitate and supernatant, respectively, obtained through immunoprecipitation of Inp with anti-HA (see Materials and Methods). M, the monomeric TGBp3:HA. (B) Examination of protein components in the IpP. P25 and P28 are the two proteins detected only in the IpP prepared from the BaMV-infected tissues (I) but not from the mock-inoculated (H) tissues. (C–E) Co-immunoprecipitation of CP, TGBp1 and TGBp2 with TGBp3:HA, respectively. C indicates purified CP, TGBp1 or TGBp2 used as reference protein for western blot. M, D, Tr and Te shown on the right margin of panel E indicate the monomeric, dimeric, trimeric and tetrameric TGBp2. (F) Analysis of endogenous viral RNA and replicase in the IpP prepared from the BaMV-infected tissues. The positions of genomic (g)RNA, subgenomic (sg)RNA 1 and sgRNA 2 on the RNA gel are shown on the left margin. C, virus replication in the presence of 1.5% NP-40 solubilized P30 fraction which contains BaMV vRNA and replicase.
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ppat-1003405-g004: Co-immunoprecipitation of various virus components with TGBp3:HA.(A) Immunoprecipitation of the TGBp3:HA-containing membrane protein complex. Inp, the S100 prepared from mock-inoculated (H) and BaMV-infected tissues (I) pre-cleaned with Protein A-Sepharose (PA). IpP and IpS, the immunoprecipitate and supernatant, respectively, obtained through immunoprecipitation of Inp with anti-HA (see Materials and Methods). M, the monomeric TGBp3:HA. (B) Examination of protein components in the IpP. P25 and P28 are the two proteins detected only in the IpP prepared from the BaMV-infected tissues (I) but not from the mock-inoculated (H) tissues. (C–E) Co-immunoprecipitation of CP, TGBp1 and TGBp2 with TGBp3:HA, respectively. C indicates purified CP, TGBp1 or TGBp2 used as reference protein for western blot. M, D, Tr and Te shown on the right margin of panel E indicate the monomeric, dimeric, trimeric and tetrameric TGBp2. (F) Analysis of endogenous viral RNA and replicase in the IpP prepared from the BaMV-infected tissues. The positions of genomic (g)RNA, subgenomic (sg)RNA 1 and sgRNA 2 on the RNA gel are shown on the left margin. C, virus replication in the presence of 1.5% NP-40 solubilized P30 fraction which contains BaMV vRNA and replicase.

Mentions: To identify the proteins associated with the Sarkosyl-extracted TGBp3:HA-based protein complex, immunoprecipitation of TGBp3:HA in the complex with anti-HA was first performed (Figure 4A). To begin with, the S100 samples (lanes 3 and 4) prepared from the healthy (H) and BaMV-infected (I) tissues were pre-cleaned with Protein A-Sepharose to generate Inp, the sample used for immunoprecipitation (lanes 5 and 6). After immunoprecipitation of the Inp with anti-HA, a significant portion of TGBp3:HA was found to reside in both IpP, the immunoprecipitate (lane 8) and IpS, the supernatant (lane 10). However, the cross-reacting signal (as indicated by *) migrating more slowly than the 31-kDa marker protein remained in IpS (lanes 9 and 10). These results indicated that the immunoprecipitation is specific for TGBp3:HA. Therefore, we further examined the proteins specifically co-immunoprecipitated with TGBp3:HA (IpP) by silver staining after Tricine SDS-PAGE (Figure 4B). Two proteins, P28 and P25 (lanes 6, 8 and 10), absent from the immunoprecipitate prepared in parallel from healthy leaves (lanes 5, 7, and 9), were detected. Both proteins were recovered from the gel, subjected to LC-MS/MS analysis (see Text S1) and identified to be TGBp1 and CP, which are essential for BaMV movement. To see whether there were other viral components associated with the TGBp3-based complex, the IpP was further subjected to western blotting (Figures 4C–E) and RdRp assay (Figure 4F). In western blotting, CP (Figure 4C, lane 8), TGBp1 (Figure 4D, lane 8) and TGBp2 (Figure 4E, lane 8) were detected. The detection of CP and TGBp1 was consistent with data obtained from LC-MS/MS analysis. In RdRp assay (Figure 4F), similar patterns of RNA transcripts were observed for replication with IpP prepared from infected tissues and with positive control (C), the 1.5% NP-40 solubilized P30 fraction containing BaMV replicase and endogenous vRNA [41]. These results indicated that there are endogenous vRNA and replicase in the IpP. Taken together, our results suggested that TGBp2, TGBp1, CP, vRNA and replicase are associated with the TGBp3-based complex.


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-immunoprecipitation of various virus components with TGBp3:HA.(A) Immunoprecipitation of the TGBp3:HA-containing membrane protein complex. Inp, the S100 prepared from mock-inoculated (H) and BaMV-infected tissues (I) pre-cleaned with Protein A-Sepharose (PA). IpP and IpS, the immunoprecipitate and supernatant, respectively, obtained through immunoprecipitation of Inp with anti-HA (see Materials and Methods). M, the monomeric TGBp3:HA. (B) Examination of protein components in the IpP. P25 and P28 are the two proteins detected only in the IpP prepared from the BaMV-infected tissues (I) but not from the mock-inoculated (H) tissues. (C–E) Co-immunoprecipitation of CP, TGBp1 and TGBp2 with TGBp3:HA, respectively. C indicates purified CP, TGBp1 or TGBp2 used as reference protein for western blot. M, D, Tr and Te shown on the right margin of panel E indicate the monomeric, dimeric, trimeric and tetrameric TGBp2. (F) Analysis of endogenous viral RNA and replicase in the IpP prepared from the BaMV-infected tissues. The positions of genomic (g)RNA, subgenomic (sg)RNA 1 and sgRNA 2 on the RNA gel are shown on the left margin. C, virus replication in the presence of 1.5% NP-40 solubilized P30 fraction which contains BaMV vRNA and replicase.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003405-g004: Co-immunoprecipitation of various virus components with TGBp3:HA.(A) Immunoprecipitation of the TGBp3:HA-containing membrane protein complex. Inp, the S100 prepared from mock-inoculated (H) and BaMV-infected tissues (I) pre-cleaned with Protein A-Sepharose (PA). IpP and IpS, the immunoprecipitate and supernatant, respectively, obtained through immunoprecipitation of Inp with anti-HA (see Materials and Methods). M, the monomeric TGBp3:HA. (B) Examination of protein components in the IpP. P25 and P28 are the two proteins detected only in the IpP prepared from the BaMV-infected tissues (I) but not from the mock-inoculated (H) tissues. (C–E) Co-immunoprecipitation of CP, TGBp1 and TGBp2 with TGBp3:HA, respectively. C indicates purified CP, TGBp1 or TGBp2 used as reference protein for western blot. M, D, Tr and Te shown on the right margin of panel E indicate the monomeric, dimeric, trimeric and tetrameric TGBp2. (F) Analysis of endogenous viral RNA and replicase in the IpP prepared from the BaMV-infected tissues. The positions of genomic (g)RNA, subgenomic (sg)RNA 1 and sgRNA 2 on the RNA gel are shown on the left margin. C, virus replication in the presence of 1.5% NP-40 solubilized P30 fraction which contains BaMV vRNA and replicase.
Mentions: To identify the proteins associated with the Sarkosyl-extracted TGBp3:HA-based protein complex, immunoprecipitation of TGBp3:HA in the complex with anti-HA was first performed (Figure 4A). To begin with, the S100 samples (lanes 3 and 4) prepared from the healthy (H) and BaMV-infected (I) tissues were pre-cleaned with Protein A-Sepharose to generate Inp, the sample used for immunoprecipitation (lanes 5 and 6). After immunoprecipitation of the Inp with anti-HA, a significant portion of TGBp3:HA was found to reside in both IpP, the immunoprecipitate (lane 8) and IpS, the supernatant (lane 10). However, the cross-reacting signal (as indicated by *) migrating more slowly than the 31-kDa marker protein remained in IpS (lanes 9 and 10). These results indicated that the immunoprecipitation is specific for TGBp3:HA. Therefore, we further examined the proteins specifically co-immunoprecipitated with TGBp3:HA (IpP) by silver staining after Tricine SDS-PAGE (Figure 4B). Two proteins, P28 and P25 (lanes 6, 8 and 10), absent from the immunoprecipitate prepared in parallel from healthy leaves (lanes 5, 7, and 9), were detected. Both proteins were recovered from the gel, subjected to LC-MS/MS analysis (see Text S1) and identified to be TGBp1 and CP, which are essential for BaMV movement. To see whether there were other viral components associated with the TGBp3-based complex, the IpP was further subjected to western blotting (Figures 4C–E) and RdRp assay (Figure 4F). In western blotting, CP (Figure 4C, lane 8), TGBp1 (Figure 4D, lane 8) and TGBp2 (Figure 4E, lane 8) were detected. The detection of CP and TGBp1 was consistent with data obtained from LC-MS/MS analysis. In RdRp assay (Figure 4F), similar patterns of RNA transcripts were observed for replication with IpP prepared from infected tissues and with positive control (C), the 1.5% NP-40 solubilized P30 fraction containing BaMV replicase and endogenous vRNA [41]. These results indicated that there are endogenous vRNA and replicase in the IpP. Taken together, our results suggested that TGBp2, TGBp1, CP, vRNA and replicase are associated with the TGBp3-based complex.

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