Rebelo Ana Rita, Niewiadomski Stella, Prosser Sean W, Krell Peter, Meng Baozhong
Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Virus Res. 2008 Dec;138(1-2):57-69. doi: 10.1016/j.virusres.2008.08.011. Epub 2008 Oct 4.
Grapevine rupestris stem pitting-associated virus (GRSPaV; Foveavirus; Flexiviridae) contains a positive-sense, ssRNA genome. GRSPaV occurs worldwide in grapes and is involved in the Rugose Wood disease complex. The GRSPaV genome contains the triple gene block (TGB), a genetic module present in several genera of plant RNA viruses. TGB encodes three proteins (TGBp1, TGBp2 and TGBp3) that are believed to work together to achieve intra- and inter-cellular transport of virions in infected plants. To reveal the subcellular localization of each TGB protein and to examine the impact that different fusion positions may have on the behavior of the native protein, we made a series of expression constructs and expressed the corresponding protein fusions in Nicotiana tabacum BY-2 cells and protoplasts. We demonstrated that TGBp1 had both a cytosolic and nuclear distribution. Two TGBp1 fusions (GFP fused at the N- or C-terminus) differ in subcellular distribution. Through the use of truncation mutants, we mapped TGBp1 regions responsible for the formation of two distinct types of aggregates. Sequence analyses predicted two and one transmembrane domains in TGBp2 and TGBp3, respectively. GFP fusions at either terminus of TGBp2 revealed identical localization to the ER network and ER-derived structures. In contrast, the two TGBp3 fusions to mRFP differed in localization. This is the first report on the subcellular localization of the viral proteins of a member of the Foveavirus genus.
沙地葡萄茎痘相关病毒（GRSPaV；凹顶病毒属；柔线病毒科）含有一条正义单链RNA基因组。GRSPaV在全球范围内的葡萄中均有出现，并与皱木复合病有关。GRSPaV基因组包含三基因块（TGB），这是一种存在于几种植物RNA病毒属中的遗传模块。TGB编码三种蛋白质（TGBp1、TGBp2和TGBp3），据信它们共同作用以实现病毒粒子在受感染植物中的细胞内和细胞间运输。为了揭示每种TGB蛋白的亚细胞定位，并研究不同融合位置可能对天然蛋白行为产生的影响，我们构建了一系列表达载体，并在烟草BY-2细胞和原生质体中表达了相应的蛋白融合体。我们证明TGBp1在细胞质和细胞核中均有分布。两种TGBp1融合体（GFP分别融合在N端或C端）在亚细胞分布上有所不同。通过使用截短突变体，我们确定了负责形成两种不同类型聚集体的TGBp1区域。序列分析预测TGBp2和TGBp3分别有两个和一个跨膜结构域。TGBp2两端的GFP融合体显示出与内质网网络和内质网衍生结构相同的定位。相比之下，两种与mRFP融合的TGBp3在定位上有所不同。这是关于凹顶病毒属成员病毒蛋白亚细胞定位的首次报道。
