Johnson Kristen A, Pokhrel Rudramani, Budicini Melissa R, Gerstman Bernard S, Chapagain Prem P, Stahelin Robert V
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Physics Department, Florida International University, Miami, FL 33199, USA.
Pathogens. 2020 May 22;9(5):402. doi: 10.3390/pathogens9050402.
The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp in eVP40, we prepared mutations of Trp (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.
埃博拉病毒（EBOV）含有七个基因，其中之一是基质蛋白eVP40，它是一种外周蛋白，足以诱导宿主细胞质膜形成病毒样颗粒。eVP40在病毒生命周期中可形成不同结构以履行不同功能，尽管促使其二聚体、六聚体和八聚体形成的结构动力学仍知之甚少。eVP40在第95位和第191位有两个保守的色氨酸残基。色氨酸的作用已得到深入研究，因为它是eVP40寡聚体形成中的一个重要残基。为深入了解色氨酸在eVP40中的功能作用，我们制备了色氨酸突变体（W191A或W191F），以确定突变对eVP40质膜定位、出芽以及eVP40寡聚化的影响。这些体外和细胞实验通过对野生型（WT）eVP40结构与W191A结构的分子动力学模拟得到补充。综合来看，色氨酸在第191位显示为关键氨基酸，因为突变为丙氨酸会降低VP40定位于质膜内小叶并形成新病毒样颗粒的能力。此外，色氨酸突变为丙氨酸或苯丙氨酸会通过破坏色氨酸与附近残基的相互作用，使体外平衡向八聚体形式转变。这项研究为eVP40结构稳定性中结构域间相互作用的重要性以及eVP40寡聚化对于质膜定位和病毒出芽的关键时机提供了新的见解。
