Gould Joseph R, Qiu Shihong, Shang Qiao, Dokland Terje, Ogino Tomoaki, Petit Chad M, Green Todd J
Department of Microbiology, University of Alabama at Birmingham.
Department of Medical Microbiology and Immunology, University of Toledo.
J Virol. 2021 Mar 10;95(7). doi: 10.1128/JVI.02180-20. Epub 2021 Jan 13.
Vesicular stomatitis virus (VSV) is a member of the order , which consists of viruses with a genome of nonsegmented negative-sense (NNS) RNA. Many insights into the molecular biology of NNS viruses were first made in VSV, which is often studied as a prototype for members of this order. Like other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and phosphoprotein (P). Recent discoveries have produced a model in which the N-terminal disordered segment of P (P) coordinates the C-terminal accessory domains to produce a "compacted" L conformation. Despite this advancement, the role of the three phosphorylation sites in P has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between P and the L protein C-terminal domain (L), we demonstrated our ability to sensitively test for changes in the interface between the two proteins. This method showed that the binding site for P on L is longer than was previously appreciated. We demonstrated that phosphorylation of P modulates its interaction with L and used a minigenome reporter system to validate the functional significance of the P-L interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated P displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of P modulates its cofactor and conformational regulatory activities with L. Polymerase-cofactor interactions like those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Despite cofactor phosphorylation being present in most of these interactions, what effect if any it has on this protein-protein interaction had not been addressed. Our study is the first to address the effects of phosphorylation on P during its interactions with L in residue-by-residue detail. As phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggest that the structural picture of L during infection might be more complex than previously appreciated.
水泡性口炎病毒（VSV）是 目 的成员，该目由具有非节段负链（NNS）RNA 基因组的病毒组成。对 NNS 病毒分子生物学的许多见解最初是在 VSV 中获得的，VSV 常被作为该目成员的原型进行研究。与其他 NNS 病毒一样，VSV RNA 聚合酶由大蛋白（L）和磷蛋白（P）组成的复合物构成。最近的发现产生了一个模型，其中 P 的 N 端无序片段（P ）协调 C 端辅助结构域以产生“紧凑”的 L 构象。尽管有这一进展，但 P 中三个磷酸化位点的作用仍不清楚。利用核磁共振光谱分析 P 与 L 蛋白 C 端结构域（L ）之间的相互作用，我们证明了我们能够灵敏地检测这两种蛋白之间界面的变化。该方法表明 P 在 L 上的结合位点比以前认为的要长。我们证明 P 的磷酸化调节其与 L 的相互作用，并使用微型基因组报告系统验证 P-L 相互作用的功能意义。使用电子显微镜方法，我们表明与磷酸化 P 结合的 L 在溶液中显示出增加的构象异质性。总体而言，我们的研究提出了一个模型，其中 P 的磷酸化调节其与 L 的辅因子和构象调节活性。像本研究中涉及的聚合酶-辅因子相互作用是单股负链 RNA 病毒 RNA 合成的绝对要求。尽管在大多数这些相互作用中存在辅因子磷酸化，但它对这种蛋白质-蛋白质相互作用有什么影响（如果有的话）尚未得到解决。我们的研究首次逐残基详细研究了磷酸化对 P 与 L 相互作用期间 P 的影响。由于磷酸化是辅因子的生物学相关状态，我们对其对 L 构象影响的证明表明，感染期间 L 的结构图景可能比以前认为的更复杂。
