麻疹病毒磷蛋白X结构域与大型聚合酶蛋白的二分界面可调节病毒聚合酶的动力学。,PLoS Pathogens

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麻疹病毒磷蛋白X结构域与大型聚合酶蛋白的二分界面可调节病毒聚合酶的动力学。
PLoS Pathogens ( IF 5.5 ) Pub Date : 2019-08-05 , DOI: 10.1371/journal.ppat.1007995
Venice Du Pont ₁ , Yi Jiang ₂ , Richard K Plemper ₁

Affiliation

麻疹病毒（MeV）是一种具有高度传染性的，重新出现的主要人类病原体。复制需要一种病毒RNA依赖性RNA聚合酶（RdRP），该酶由与同四聚体磷蛋白（P）复合的大（L）聚合酶蛋白组成。另外，P介导与核蛋白（N）衣壳化的病毒RNA基因组的相互作用。对核糖核蛋白模板的P：L接口和RdRP协商的性质了解甚少。基于生化界面映射，针对酵母GCN4的中央P四聚结构域（OD）交换和功能测定，我们证明MeV P-L界面是两部分的，包括靠近OD的卷曲螺旋微结构域和三角棱柱形C端P X域（P-XD）的空表面，与绑定N尾的已知P-XD表面不同。混合的无效突变体P四聚体以比例依赖的方式恢复了L结合能力，并在小型复制子测定和重组MeV中完全恢复了生物活性，这表明各个L结合界面元素在物理上和机理上都是不同的。对L和N的P-XD结合能力同样是反互补的，结合数学模型，可以通过化学计量控制的三向和三向互补作用对P进行机械表征。每个P四聚体的四个XD中只有一个必须具有L或N结合能力才能具有生物活性，但是同一P-XD与L和N的相互作用是互斥的，并且L结合取代了N。，指定为具有L结合能力的P-XD会导致明显的RdRP过度活跃，

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Bipartite interface of the measles virus phosphoprotein X domain with the large polymerase protein regulates viral polymerase dynamics.

Measles virus (MeV) is a highly contagious, re-emerging, major human pathogen. Replication requires a viral RNA-dependent RNA polymerase (RdRP) consisting of the large (L) polymerase protein complexed with the homo-tetrameric phosphoprotein (P). In addition, P mediates interaction with the nucleoprotein (N)-encapsidated viral RNA genome. The nature of the P:L interface and RdRP negotiation of the ribonucleoprotein template are poorly understood. Based on biochemical interface mapping, swapping of the central P tetramerization domain (OD) for yeast GCN4, and functional assays, we demonstrate that the MeV P-to-L interface is bipartite, comprising a coiled-coil microdomain proximal to the OD and an unoccupied face of the triangular prism-shaped C-terminal P X-domain (P-XD), which is distinct from the known P-XD face that binds N-tail. Mixed null-mutant P tetramers regained L-binding competence in a ratio-dependent manner and fully reclaimed bioactivity in minireplicon assays and recombinant MeV, demonstrating that the individual L-binding interface elements are physically and mechanistically distinct. P-XD binding competence to L and N was likewise trans-complementable, which, combined with mathematical modeling, enabled the mechanistic characterization of P through two- and stoichiometrically-controlled three-way complementations. Only one each of the four XDs per P tetramer must be L or N binding-competent for bioactivity, but interaction of the same P-XD with L and N was mutually exclusive, and L binding superseded engaging N. Mixed P tetramers with a single, designated L binding-competent P-XD caused significant RdRP hyperactivity, outlining a model of iterative resolution and reformation of the P-XD:L interface regulating polymerase mobility.

更新日期：2019-08-05

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