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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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Related in: MedlinePlus

Effects of detergent and additive on extraction of the TGBp3:HA-containing membrane protein complex from P30.The TGBp3-containing membrane protein complexes in P30 were extracted with Triton X-100 (Tx) at 26°C in the presence or absence of high pH (pH 11), NDSB and mβCD, or extracted with 1% Sarkosyl (Sarko.). P and S indicate the supernatant and pellet samples, respectively, after extraction of the P30 with detergent and ultracentrifugation at 100,000 g. C, the TGBp3:HA in P30 before extraction. M, the monomeric TGBp3:HA.
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ppat-1003405-g003: Effects of detergent and additive on extraction of the TGBp3:HA-containing membrane protein complex from P30.The TGBp3-containing membrane protein complexes in P30 were extracted with Triton X-100 (Tx) at 26°C in the presence or absence of high pH (pH 11), NDSB and mβCD, or extracted with 1% Sarkosyl (Sarko.). P and S indicate the supernatant and pellet samples, respectively, after extraction of the P30 with detergent and ultracentrifugation at 100,000 g. C, the TGBp3:HA in P30 before extraction. M, the monomeric TGBp3:HA.

Mentions: The membrane integration of TGBp3 led us to presume that extraction of the TGBp3-based complex from the ER or ER-derived membrane vesicles with a compatible detergent would enable us to isolate the virus movement complex if the TGBp3-based complex really serves as a vehicle for virus movement [29]. However, non-ionic detergent such as n-Dodecyl-β-maltoside (DDM), IGEPAL CA-630 or Triton X-100 failed to extract TGBp3-based complex from P30 (data not shown). Thus, non-detergent sulfobetaines (NDSBs) additive [36], methyl β-cyclodextrin (mβCD) [37], [38], pH or higher temperature, which may assist the extraction of membrane protein complex, was incorporated into the Triton X-100 extraction system. However, no TGBp3:HA was able to be extracted from P30 (P) to S100 (S) after treatment with higher temperature (Figure 3, lanes 3 and 4), NDSBs (lanes 5 and 6), pH (lanes 9 and 10) or mβCD (lanes 11 and 12), indicating that extraction of TGBp3-based protein complex from P30 remained inefficient after the above-mentioned treatments. The inefficiency of TGBp3 extraction, we thought, might be due to co-precipitation of the Triton X-100 extracted TGBp3-based protein complex with certain existing large complexes during ultracentrifugation, since the concentration of Triton X-100 (1%) used for the extraction was much higher than the critical micellar concentration of Triton X-100 (0.015%). To avoid the precipitation problem, an ionic detergent, Sarkosyl, used to purify BaMV RNA-capping enzyme and assay the endogenous RNA-dependent-RNA polymerase (RdRp) activity of BaMV [39], [40] was adopted. Fortunately, most of the TGBp3:HA signal or TGBp3:HA-based complex was extracted from P30 and retained in S100 after ultracentrifugation (Figure 3, lanes 7 and 8).


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)

Effects of detergent and additive on extraction of the TGBp3:HA-containing membrane protein complex from P30.The TGBp3-containing membrane protein complexes in P30 were extracted with Triton X-100 (Tx) at 26°C in the presence or absence of high pH (pH 11), NDSB and mβCD, or extracted with 1% Sarkosyl (Sarko.). P and S indicate the supernatant and pellet samples, respectively, after extraction of the P30 with detergent and ultracentrifugation at 100,000 g. C, the TGBp3:HA in P30 before extraction. M, the monomeric TGBp3:HA.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003405-g003: Effects of detergent and additive on extraction of the TGBp3:HA-containing membrane protein complex from P30.The TGBp3-containing membrane protein complexes in P30 were extracted with Triton X-100 (Tx) at 26°C in the presence or absence of high pH (pH 11), NDSB and mβCD, or extracted with 1% Sarkosyl (Sarko.). P and S indicate the supernatant and pellet samples, respectively, after extraction of the P30 with detergent and ultracentrifugation at 100,000 g. C, the TGBp3:HA in P30 before extraction. M, the monomeric TGBp3:HA.
Mentions: The membrane integration of TGBp3 led us to presume that extraction of the TGBp3-based complex from the ER or ER-derived membrane vesicles with a compatible detergent would enable us to isolate the virus movement complex if the TGBp3-based complex really serves as a vehicle for virus movement [29]. However, non-ionic detergent such as n-Dodecyl-β-maltoside (DDM), IGEPAL CA-630 or Triton X-100 failed to extract TGBp3-based complex from P30 (data not shown). Thus, non-detergent sulfobetaines (NDSBs) additive [36], methyl β-cyclodextrin (mβCD) [37], [38], pH or higher temperature, which may assist the extraction of membrane protein complex, was incorporated into the Triton X-100 extraction system. However, no TGBp3:HA was able to be extracted from P30 (P) to S100 (S) after treatment with higher temperature (Figure 3, lanes 3 and 4), NDSBs (lanes 5 and 6), pH (lanes 9 and 10) or mβCD (lanes 11 and 12), indicating that extraction of TGBp3-based protein complex from P30 remained inefficient after the above-mentioned treatments. The inefficiency of TGBp3 extraction, we thought, might be due to co-precipitation of the Triton X-100 extracted TGBp3-based protein complex with certain existing large complexes during ultracentrifugation, since the concentration of Triton X-100 (1%) used for the extraction was much higher than the critical micellar concentration of Triton X-100 (0.015%). To avoid the precipitation problem, an ionic detergent, Sarkosyl, used to purify BaMV RNA-capping enzyme and assay the endogenous RNA-dependent-RNA polymerase (RdRp) activity of BaMV [39], [40] was adopted. Fortunately, most of the TGBp3:HA signal or TGBp3:HA-based complex was extracted from P30 and retained in S100 after ultracentrifugation (Figure 3, lanes 7 and 8).

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