Department of Chemistry, Indiana University, Bloomington, IN, USA.
J Virol. 2013 Mar;87(6):3502-15. doi: 10.1128/JVI.03112-12. Epub 2013 Jan 9.
Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses that infect a variety of vertebrate hosts. The CoV nucleocapsid (N) protein contains two structurally independent RNA binding domains, designated the N-terminal domain (NTD) and the dimeric C-terminal domain (CTD), joined by a charged linker region rich in serine and arginine residues (SR-rich linker). An important goal in unraveling N function is to molecularly characterize N-protein interactions. Recent genetic evidence suggests that N interacts with nsp3a, a component of the viral replicase. Here we present the solution nuclear magnetic resonance (NMR) structure of mouse hepatitis virus (MHV) nsp3a and show, using isothermal titration calorimetry, that MHV N219, an N construct that extends into the SR-rich linker (residues 60 to 219), binds cognate nsp3a with high affinity (equilibrium association constant [K(a)], [1.4 ± 0.3] × 10(6) M(-1)). In contrast, neither N197, an N construct containing only the folded NTD (residues 60 to 197), nor the CTD dimer (residues 260 to 380) binds nsp3a with detectable affinity. This indicates that the key nsp3a binding determinants localize to the SR-rich linker, a finding consistent with those of reverse genetics studies. NMR chemical shift perturbation analysis reveals that the N-terminal region of an MHV N SR-rich linker peptide (residues 198 to 230) binds to the acidic face of MHV nsp3a containing the acidic α2 helix with an affinity (expressed as K(a)) of 8.1 × 10(3) M(-1). These studies reveal that the SR-rich linker of MHV N is necessary but not sufficient to maintain this high-affinity binding to N.
冠状病毒（CoV）是一种正链、单链、有包膜的 RNA 病毒，可感染多种脊椎动物宿主。CoV 核衣壳（N）蛋白包含两个结构上独立的 RNA 结合结构域，分别命名为 N 端结构域（NTD）和二聚体 C 端结构域（CTD），由富含丝氨酸和精氨酸残基的带电连接区（SR 富含连接区）连接。阐明 N 蛋白功能的一个重要目标是从分子水平上阐明 N 蛋白相互作用。最近的遗传证据表明，N 蛋白与病毒复制酶的一个组成部分 nsp3a 相互作用。本文报道了鼠肝炎病毒（MHV）nsp3a 的溶液核磁共振（NMR）结构，并通过等温滴定量热法表明，MHV N219（延伸到 SR 富含连接区的 N 结构域，残基 60 至 219）与同源 nsp3a 具有高亲和力（平衡结合常数 [K(a)]，[1.4 ± 0.3] × 10(6) M(-1)）。相比之下，N197（仅包含折叠 NTD 的 N 结构域，残基 60 至 197）和 CTD 二聚体（残基 260 至 380）都不能与 nsp3a 结合。这表明 nsp3a 结合的关键决定因素位于 SR 富含连接区，这一发现与反向遗传学研究的结果一致。NMR 化学位移扰动分析表明，MHV N 的 SR 富含连接区的 N 端区域（残基 198 至 230）与含有酸性 α2 螺旋的 MHV nsp3a 的酸性面结合，亲和力（以 K(a)表示）为 8.1 × 10(3) M(-1)。这些研究表明，MHV N 的 SR 富含连接区是维持与 N 高亲和力结合所必需的，但不是充分的。
