Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein
Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein
复制标题
DOI：
10.1128/jvi.00058-12
复制
发表时间：
2012-08-01
影响因子：
5.4
通讯作者：
Eleouet, Jean-Francois
中科院分区：
文献类型：
--
作者：
Galloux, Marie;Tarus, Bogdan;Eleouet, Jean-Francois
人类呼吸道合胞病毒(HRSV)基因组由一个与核蛋白(N)紧密相关的负义单链RNA组成。这种核糖核蛋白(RNP)复合体是病毒RNA依赖的RNA聚合酶复制和转录的模板。病毒聚合酶对RNP的识别涉及到磷蛋白(P)的C-末端结构域(P-CTD)与N之间的特异性相互作用，但N上的P结合区仍有待鉴定。本研究采用谷胱甘肽S转移酶下拉试验，将HRSVN(N-NTD)的N-末端核心区确定为P结合区。P-CTD的生化表征和N-NTD的分子模拟使我们能够在N上定义四个潜在的候选口袋(口袋I[PI]到PIV)作为被正电荷区域包围的疏水部位，这些部位可以构成P-CTD相互作用结构域的互补部位。选择的氨基酸在P识别N-RNA复合体中的作用首先是通过基于包含荧光素酶报告基因的HRSV微型基因组的聚合酶活性分析的定点突变来筛选的。当改变为Ala时，PI的大部分残基对病毒RNA合成是关键的，其中R132a突变体的作用最强。这些突变也减少或取消了体外和体内的P-N相互作用，如GST下拉和免疫沉淀实验所确定的。这些残基形成的口袋是P与N-RNA复合体结合的关键，是肺炎病毒N蛋白所特有的，与迄今已发现的其他非分段负链病毒的P结合位点明显不同。
The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P-CTD) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N-NTD) as a P binding domain. A biochemical characterization of the P-CTD and molecular modeling of the N-NTD, allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P-CTD interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.