Kingston Richard L, Baase Walter A, Gay Leslie S
Howard Hughes Medical Institute, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.
J Virol. 2004 Aug;78(16):8630-40. doi: 10.1128/JVI.78.16.8630-8640.2004.
We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C. Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.
我们报告了一项关于麻疹病毒和腮腺炎病毒的P蛋白与RNA相关的N蛋白（N-RNA）之间相互作用的分析，其中蛋白质是在细菌表达系统中产生的。在这项研究中，我们证实N蛋白的C末端尾巴对于核衣壳形成并非必需。对于麻疹病毒和腮腺炎病毒的N蛋白，包含氨基酸1至375的截短蛋白在细菌细胞内组装成核衣壳样颗粒。对于麻疹病毒N蛋白，P蛋白的结合位点位于分子尾部的第477至505位残基，这是麻疹病毒属中相对保守的序列。对于腮腺炎病毒N蛋白，P蛋白的结合位点位于N蛋白的组装结构域（第1至398位残基），而未检测到P蛋白与N蛋白尾部的强结合。这些结果表明，副粘病毒中聚合酶的附着位点各不相同。下拉实验表明，麻疹病毒和腮腺炎病毒P蛋白的最后50个氨基酸（麻疹病毒P蛋白，第457至507位；腮腺炎病毒P蛋白，第343至391位）自身构成核衣壳结合结构域（NBD）。光谱研究表明，两种病毒中的NBD主要为α螺旋结构。然而，只有麻疹病毒P蛋白的NBD是稳定且折叠的，腮腺炎病毒P蛋白的三级结构程度较低。通过等温滴定量热法，我们证明麻疹病毒P蛋白的NBD以1:1的化学计量比与麻疹病毒N蛋白的第477至505位残基结合。解离常数（K(d)）在20℃时测定为13μM，在37℃时为35μM。我们的数据与一个模型一致，即位于P蛋白C末端的α螺旋核衣壳结合结构域负责将病毒聚合酶拴系到其模板上，但也表明详细来说，麻疹病毒属和风疹病毒属中的聚合酶结合存在显著差异。
