Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P.
Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P.
复制标题
绘制与病毒磷蛋白 P 形成稳定复合物所需的副粘病毒 L 蛋白区域。
DOI：
10.1128/jvi.68.8.4862-4872.1994
复制
发表时间：
1994
影响因子：
5.4
通讯作者：
Parks,GD
中科院分区：
文献类型：
--
作者：
Parks,GD
副粘病毒大蛋白(L)和磷蛋白(P)都是病毒核糖核酸依赖的核糖核酸聚合酶活性所必需的。以往的生化实验表明，L和P在体内表达时可以形成复合体。本文报道了副粘病毒猴病毒5型L和P蛋白在HeLa T4细胞中的共表达，并对L-P复合体进行了检测。为了确定L-P复合体形成所需的L蛋白区域，通过突变获得了16个缺失突变体。将这些L突变体与cDNA源P共表达并标记35S氨基酸后，用免疫共沉淀法和5~20%甘油梯度沉淀法分析细胞裂解产物中稳定的L-P复合体。用这两种方法在与P的复合体中检测到L蛋白的突变形式，该突变形式含有从全长2,255个残基的C末端一半中去除多达1,008个残基的L蛋白。相反，L N端的大片段缺失导致蛋白质不能形成稳定的L-磷复合体。同样，含有较小缺失的L突变体也存在与P稳定相互作用的缺陷，这些缺失分别删除了副粘病毒和横纹病毒L蛋白之间保守的N-末端区域(结构域I、II或III)。这些结果表明，L蛋白的N-末端含有对稳定的L-P复合体形成至关重要的序列，L的C-末端并不直接参与这些相互作用。用35S氨基酸脉冲标记SV5感染的HeLa T4细胞，用梯度沉淀法检测细胞提取物。溶解的L蛋白以约8-10S的形式存在，而P蛋白则以约4S的形式存在(约占85%)，并与L共育(约占15%)。这些数据为支持非分段负义核糖核酸病毒L蛋白的简单结构提供了第一个生化证据。根据L蛋白的结构模型以及L与病毒聚合酶第二亚单位P的相互作用对结果进行了讨论。
The paramyxovirus large protein (L) and phosphoprotein (P) are both required for viral RNA-dependent RNA polymerase activity. Previous biochemical experiments have shown that L and P can form a complex when expressed from cDNA plasmids in vivo. In this report, L and P proteins of the paramyxovirus simian virus 5 (SV5) were coexpressed in HeLa T4 cells from cDNA plasmids, and L-P complexes were examined. To identify regions of the SV5 L protein that are required for L-P complex formation, 16 deletion mutants were constructed by mutagenesis of an SV5 L cDNA. Following coexpression of these L mutants with cDNA-derived P and radiolabeling with 35S-amino acids, cell lysates were analyzed for stable L-P complexes by a coimmunoprecipitation assay and by sedimentation on 5 to 20% glycerol gradients. Mutant forms of L containing deletions that removed as much as 1,008 residues from the C-terminal half of the full-length 2,255-residue L protein were detected in complexes with P by these two assays. In contrast, large deletions in the N-terminal half of L resulted in proteins that were defective in the formation of stable L-P complexes. Likewise, L mutants containing smaller deletions that individually removed N-terminal regions which are conserved among paramyxovirus and rhabdovirus L proteins (domain I, II, or III) were also defective in stable interactions with P. These results suggest that the N-terminal half of the L protein contains sequences important for stable L-P complex formation and that the C-terminal half of L is not directly involved in these interactions. SV5-infected HeLa T4 cells were pulse-labeled with 35S-amino acids, and cell extracts were examined by gradient sedimentation. Solubilized L protein was detected as an approximately 8 to 10S species, while the P protein was found as both a approximately 4S form (approximately 85%) and a species that cosedimented with L (approximately 15%). These data provide the first biochemical evidence in support of a simple domain structure for an L protein of the nonsegmented negative-sense RNA viruses. The results are discussed in terms of a structural model for the L protein and the interactions of L with the second viral polymerase subunit P.