Importance of MHV-CoV A59 nucleocapsid protein COOH-terminal negative charges.
Importance of MHV-CoV A59 nucleocapsid protein COOH-terminal negative charges.
复制标题
DOI：
10.1007/978-0-387-33012-9_22
复制
发表时间：
2006
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
1.导言全长约26-30kb，是所有RNA病毒中最大的。所有冠状病毒都至少含有四种结构蛋白：三种囊膜蛋白、膜蛋白(M)、棘蛋白(S)和囊膜蛋白(E)，以及一种50-60 kDa的磷酸化核蛋白(N)。1所有冠状病毒的N蛋白范围在375到455个氨基酸之间，并且是磷酸化的。N蛋白是一种多功能病毒基因产物。在病毒感染的细胞中，N蛋白与基因组RNA结合形成螺旋核糖核蛋白(RNP)复合体。N蛋白在转录和/或复制中也发挥着尚未确定的作用(S)，可能还在翻译控制中发挥作用。N是一种高度碱性的蛋白质，含有大量潜在的磷酸化位点。该蛋白质含有高浓度的丝氨酸残基(7-11%)。N由三个保守的结构域组成，其中两个是碱性结构域，一个是羧基末端结构域，是酸性的。2、3蛋白质的羧基末端结构域III含有许多保守的带负电荷的氨基酸。这些残基以前被假设在组装过程中与NM蛋白的相互作用中发挥作用。4-6残基也可以作为蛋白质总体功能结构的贡献者。可以想象，这些残基对病毒生命周期中蛋白质提供的任何功能都可能是重要的。作为我们了解结构域III带电残基功能重要性的目标的一部分，我们使用小鼠肝炎冠状病毒(MHV-CoV A59)感染性克隆在病毒基因组的背景下制作了一系列N突变体并进行了研究。我们发现天冬氨酸(D)440和441具有重要的功能。当任一残基单独改变为带正电荷的精氨酸(R)时，活病毒被恢复，但当两个残基都改变为丙氨酸(A)时，则不能恢复。对来自带电单突变体和中性双突变体的大量空斑纯化病毒的分析表明，除了在440和/或441位引入的突变外，几乎所有的病毒都在N基因内发生了新的氨基酸变化。所有这些补偿性变化主要集中在结构域III的氨基末端更远的一个区域。
1. INTRODUCTION approximately 26–30 kb in length, the largest of all the RNA viruses. All coronoviruses contain at least four structural proteins: three envelope proteins, the membrane (M), spike (S), and envelope (E) proteins, and a 50–60 kDa phosphorylated nucleocapsid (N) protein. 1 The N proteins of all coronaviruses range between 375 and 455 amino acids and are phosphorylated. N protein is a multifunctional viral gene product. In virus-infected cells, N protein binds to the genomic RNA to form a helical ribonucleoprotein (RNP) complex. The N protein also plays a yet undetermined role (s) in transcription and/or replication, and possibly in translational control. N is a highly basic protein that contains a large number of potential phosphorylation sites. The protein has a high concentration of serine residues (7–11%). N consists of three conserved structural domains, two are basic and one, the carboxy terminal domain, is acidic. 2, 3 A number of conserved negatively charged amino acids are located in the carboxy-terminal domain III of the protein. These residues were previously hypothesized to play a role in NM protein interactions during assembly. 4-6 The residues could alternatively serve as contributors to the general overall functional structure of the protein. Conceivably, the residues could be important for any of the functions that the protein provides during the virus life cycle. As part of our goal to understand the functional importance of the charged residues in domain III, a series of N mutants were made and studied in the context of the viral genome using a mouse hepatitis coronavirus (MHV-CoV A59) infectious clone. We found that aspartic acids (D) 440 and 441 are functionally important. Viable viruses were recovered when either residue was changed singly to positively charged arginine (R) but not when both residues were changed to alanine (A). Analysis of a large number of plaque purified viruses from the panel of charged single and neutral double mutants revealed that, in addition to the introduced mutations at positions 440 and/or 441, nearly all had new amino acid changes within the N gene. All of these compensating changes were concentrated primarily in one region further toward the amino end of domain III. A