Structure of Recombinant Rabies Virus Nucleoprotein-RNA Complex and Identification of the Phosphoprotein Binding site
Structure of Recombinant Rabies Virus Nucleoprotein-RNA Complex and Identification of the Phosphoprotein Binding site
复制标题
重组狂犬病病毒核蛋白-RNA复合物的结构及磷蛋白结合位点的鉴定
作者：
G. Schoehn;F. Iseni;M. Mavrakis;D. Blondel;R. Ruigrok
摘要 狂犬病病毒核蛋白 (N) 在昆虫细胞中产生，在其中形成核蛋白-RNA (N-RNA) 复合物，该复合物在生化和生物物理上与狂犬病病毒 N-RNA 没有区别。我们选择了与短昆虫细胞RNA结合的重组N-RNA复合物，这些RNA形成含有9至11个N单体的小环。我们还生产了重组 N-RNA 环和病毒 N-RNA，它们经过胰蛋白酶处理，并且丢失了四分之一的核蛋白 C 端。胰蛋白酶处理的 N-RNA 不再与重组狂犬病病毒磷蛋白（病毒聚合酶辅因子）结合，因此 N 的 C 末端部分的存在是磷蛋白结合所必需的。使用冷冻电子显微镜分析完整的和胰蛋白酶处理的重组 N-RNA 环，并通过单颗粒图像分析结合反投影计算三维模型。核蛋白具有双叶形状，每个单体都有两个与每个相邻单体相互作用的位点。胰蛋白酶处理会切除其中一个叶的一部分，但不会缩短蛋白质或改变其他结构参数。使用负染色电子显微镜，我们观察到与 N-RNA 环尖端结合的磷蛋白，很可能位于可以被胰蛋白酶去除的位点。基于此处确定的 N 的形状以及来自电子显微镜的游离狂犬病病毒 N-RNA 和病毒核衣壳的结构参数，我们提出了病毒中狂犬病病毒 N-RNA 的低分辨率模型。
ABSTRACT Rabies virus nucleoprotein (N) was produced in insect cells, in which it forms nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies virus N-RNA and from nucleocapsid in virus, we propose a low-resolution model for rabies virus N-RNA in the virus.
DOI：
10.1073/pnas.78.10.6191
发表时间：
1981-10
影响因子：
11.1
作者：
J. Keene;B. J. Thornton;S. Emerson
通讯作者：
J. Keene;B. J. Thornton;S. Emerson
影响因子：
3.7
作者：
FU, ZF;ZHENG, YM;DIETZSCHOLD, B
通讯作者：
DIETZSCHOLD, B
影响因子：
3
作者：
Frank, J;Radermacher, M;Leith, A
通讯作者：
Leith, A