Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins.
Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins.
复制标题
痘苗病毒 A33R 和 A36R 包膜蛋白胞质域内相互作用位点的定位和功能分析。
DOI：
10.1128/jvi.77.7.4113-4126.2003
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发表时间：
2003
影响因子：
5.4
通讯作者：
Moss,Bernard
中科院分区：
文献类型：
--
作者：
Ward,BrianM;Weisberg,AndreaS;Moss,Bernard
痘苗病毒A36R蛋白能否掺入细胞内被膜病毒粒子(IEV)的外膜依赖于A33R蛋白的表达。用酵母双杂交系统研究了A36R蛋白200个氨基酸的胞质结构域与自身或与A33R、A34R、B5R或F12L IEV膜蛋白胞质结构域的可能相互作用。仅在A36R和A33R蛋白的胞质结构域之间检测到强相互作用。经进一步分析，相互作用位点被定位在A36R蛋白的91到111个残基。为探讨A36R：A33R相互作用在病毒感染中的作用，构建了5种以B5R-GFP为标志的重组痘苗病毒。其中4株全长A36R基因被不同长度的A36R C端截短所取代，其中2株含有91~111位残基，2株缺失该区域。第五个重组病毒含有A33R基因，其胞质尾部40个氨基酸的大部分缺失。在病毒感染过程中，A36R的91~111位残基和A33R的胞质尾部是两种蛋白相互作用和在IEV上获得最大数量A36R蛋白所必需的。缺乏A33R或A36R这些区域的突变体形成的IEV只表现出短暂的零星细胞内运动，没有肌动蛋白尾巴，并在细胞单层上形成与A36R缺失突变体相同的小斑块，并且比通过特异性消除肌动蛋白尾部形成的点突变形成的斑块更小。A36R蛋白的A33R相互作用位点在正痘病毒中高度保守，并可能与微管运动和肌动蛋白尾部形成所需的细胞蛋白的结合位点重叠。
Incorporation of the vaccinia virus A36R protein into the outer membrane of intracellular enveloped virions (IEV) is dependent on expression of the A33R protein. Possible interactions of the 200-amino-acid cytoplasmic domain of the A36R protein with itself or with the cytoplasmic domain of the A33R, A34R, B5R, or F12L IEV membrane protein was investigated by using the yeast two-hybrid system. A strong interaction was detected only between the cytoplasmic domains of the A36R and A33R proteins. Upon further analyses, the interaction site was mapped to residues 91 to 111 of the A36R protein. To investigate the role of the A36R:A33R interaction during viral infection, five recombinant vaccinia viruses containing B5R-GFP as a marker were constructed. Four had the full-length A36R gene replaced with various-length C-terminal truncations of A36R, of which two contained residues 91 to 111 and two were missing this region. The fifth recombinant virus had an A33R gene with most of the 40-amino-acid cytoplasmic tail deleted. Residues 91 to 111 of A36R and the cytoplasmic tail of A33R were required for a strong interaction between the two proteins during viral infection and for maximal amounts of A36R protein on IEV. Mutants lacking these regions of A33R or A36R formed IEV that exhibited only short sporadic intracellular movement, displayed no actin tails, and formed small plaques on cell monolayers equivalent to those of an A36R deletion mutant and smaller than those formed by point mutations that specifically abrogate actin tail formation. The A33R interaction site of the A36R protein is highly conserved among orthopoxviruses and may overlap binding sites for cellular proteins needed for microtubular movement and actin tail formation.