The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.
The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.
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DOI：
10.1371/journal.ppat.1003405
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发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Chang BY
中科院分区：
文献类型：
--
作者：
Chou YL;Hung YJ;Tseng YH;Hsu HT;Yang JY;Wung CH;Lin NS;Meng M;Hsu YH;Chang BY
竹叶花叶病毒（BaMV）的三基因阻断蛋白3 （TGBp3）是一种完整的内质网（ER）膜蛋白，被认为是形成膜复合物以在细胞内传递病毒。然而，传递到胞间连丝（PD）的病毒实体及其与tgbp3基复合物的关联尚不清楚。TGBp3在膜泡中的化学提取和部分蛋白水解结果表明，TGBp3具有右向外的膜拓扑结构；即TGBp3的c端尾部暴露于内质网外表面。对萨科齐法提取的TGBp3复合物的TGBp3特异性免疫沉淀分析表明，TGBp1、TGBp2、TGBp3、衣壳蛋白（CP）、复制酶和病毒RNA是病毒运动复合物的潜在组分。在TGBp3特异性免疫沉淀后检测到病毒粒子的早期洗脱凝胶过滤组分中，TGBp2、TGBp3和CP（而不是TGBp1）的大量共分离表明，TGBp2和TGBp3复合物能够稳定地与病毒粒子结合。这一概念被纯化病毒粒子的免疫金标记透射电子显微镜（TEM）证实。此外，突变和共聚焦显微镜分析显示，TGBp3通过增强TGBp1依赖于TGBp2和TGBp3的PD定位，在病毒细胞间运动中起关键作用。综上所述，我们的研究结果表明，痘病毒的细胞间运动需要病毒粒子与TGBp2和tgbp3基膜复合物稳定结合，并通过该复合物将TGBp1募集到PD中。植物病毒通过病毒编码的运动蛋白和宿主因子，通过胞间连丝（plasmodesmata， PD）在细胞间传播其感染实体。一些RNA病毒编码三种功能协调的运动蛋白，组织成三重基因块（TGB），以促进它们的细胞间运动。已知TGBp2和TGBp3与内质网（ER）膜和ER源性囊泡有关。内质网或囊泡相关的TGBp2和TGBp3可能会形成一个膜复合物来传递病毒。然而，对于potexlike病毒，“病毒RNA货物”的身份以及货物是否能够在细胞内运输过程中与TGBp2-和tgbp3 -含膜复合物结合仍不清楚。利用ha标记和his标记的竹花叶病毒（BaMV） TGBp3结构，我们能够确定TGBp3的膜拓扑结构，分离TGBp3复合物，并检测稳定的tgbp2 -TGBp3病毒粒子复合物的存在。此外，我们已经明确了TGBp3通过增强TGBp1的TGBp2和TGBp3依赖性PD定位在病毒细胞间运动中起关键作用。这些结果表明，痘病毒的细胞间运动需要病毒粒子与含TGBp2-和tgbp3的膜复合体稳定结合，并通过该复合体将TGBp1招募到PD。
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. Plant viruses spread their infectious entities from cell to cell via plasmodesmata (PD) through the assistance of virus-encoded movement proteins and host factors. Some RNA viruses encode three functionally coordinated movement proteins organized into a triple gene block (TGB) to facilitate their cell-to-cell movement. TGBp2 and TGBp3 are known to associate with the endoplasmic reticulum (ER) membrane and ER-derived vesicles. The ER- or vesicle-associated TGBp2 and TGBp3 presumably form a membrane complex to deliver the viruses. However, the identity of the “viral RNA cargo” and whether the cargo is able to associate with the TGBp2- and TGBp3-containing membrane complex during intracellular transport remain unclear for potex-like viruses. Taking advantage of an HA-tagged and a His-tagged TGBp3 construct of Bamboo mosaic virus (BaMV), we have been able to determine the membrane topology of TGBp3, isolate the TGBp3-based complex and detect the existence of a stable TGBp2-TGBp3-virion complex. Moreover, we have clarified that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. These results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-containing membrane complex and recruitment of TGBp1 to the PD by this complex.
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DOI：
10.1083/jcb.93.1.97
发表时间：
1982-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Fujiki Y;Hubbard AL;Fowler S;Lazarow PB
通讯作者：
Lazarow PB
影响因子：
11.6
作者：
Haupt, S;Cowan, GH;Torrance, L
通讯作者：
Torrance, L
影响因子：
3.7
作者：
Bamunusinghe, Devinka;Hemenway, Cynthia L.;Verchot-Lubicz, Jeanmarie
通讯作者：
Verchot-Lubicz, Jeanmarie
影响因子：
7.2
作者：
Bayne, EH;Rakitina, DV;Baulcombe, DC
通讯作者：
Baulcombe, DC
影响因子：
3.8
作者：
LIN, NS;LIN, BY;HSU, YH
通讯作者：
HSU, YH