Ward B M, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0445, USA.
J Virol. 2000 Apr;74(8):3771-80. doi: 10.1128/jvi.74.8.3771-3780.2000.
The vaccinia virus B5R type I integral membrane protein accumulates in the Golgi network, from where it becomes incorporated into the envelope of extracellular virions. Our objective was to determine the domains of B5R responsible for Golgi membrane targeting in the absence of other viral components. Fusion of an enhanced green fluorescent protein to the C terminus of B5R allowed imaging of the chimeric protein without altering intracellular trafficking and Golgi network localization in transfected cells. Deletion or swapping of B5R domains with corresponding regions of the vesicular stomatitis virus G protein, which is targeted to the plasma membrane, indicated that (i) the N-terminal extracellular domain of B5R had no specific role in Golgi apparatus localization, (ii) the transmembrane domain of B5R was sufficient for exiting the endoplasmic reticulum, and (iii) removal of the cytoplasmic tail impaired Golgi network localization and increased the accumulation of B5R in the plasma membrane. Further experiments demonstrated that the cytoplasmic tail mediated internalization of B5R from the plasma membrane, suggesting a retrieval mechanism. Mutagenesis revealed residues required for Golgi membrane localization and efficient plasma membrane retrieval of the B5R protein: a tyrosine at residue 310 and two adjacent leucines at residues 315 and 316.
痘苗病毒B5R I型整合膜蛋白在高尔基体网络中积累，随后从该部位被整合到细胞外病毒粒子的包膜中。我们的目标是确定在没有其他病毒成分的情况下，B5R中负责靶向高尔基体膜的结构域。将增强型绿色荧光蛋白与B5R的C末端融合，使得在不改变转染细胞内吞运输和高尔基体网络定位的情况下，能够对嵌合蛋白进行成像。用靶向质膜的水泡性口炎病毒G蛋白的相应区域对B5R结构域进行缺失或交换，结果表明：（i）B5R的N末端胞外结构域在高尔基体定位中没有特定作用；（ii）B5R的跨膜结构域足以使其从内质网中输出；（iii）去除细胞质尾巴会损害高尔基体网络定位，并增加B5R在质膜中的积累。进一步的实验表明，细胞质尾巴介导了B5R从质膜的内化，提示存在一种回收机制。诱变揭示了B5R蛋白高尔基体膜定位和质膜有效回收所需的残基：310位的酪氨酸以及315和316位的两个相邻亮氨酸。
