Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
J Mol Biol. 2010 Jun 11;399(3):347-57. doi: 10.1016/j.jmb.2010.04.022. Epub 2010 Apr 24.
Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.
埃博拉病毒是导致人类和非人类灵长类动物致命性出血热的病原体。在迄今为止所描述的丝状病毒中，雷斯顿埃博拉病毒（REBOV）是唯一一种对人类无致病性的埃博拉病毒，尽管 REBOV 可导致非人类灵长类动物致命疾病。先前的研究还表明，REBOV 在抑制宿主固有免疫反应方面的效果不如扎伊尔埃博拉病毒（ZEBOV）或马尔堡病毒。病毒编码的 VP35 蛋白对于免疫抑制至关重要，但目前对于 REBOV 和其他丝状病毒的 VP35 蛋白的相对贡献尚不清楚。为了解决这个问题，我们使用结构、生化和病毒学研究方法对 REBOV VP35 干扰素抑制结构域（IID）进行了表征。这些研究揭示了两种病毒在双链 RNA 结合和干扰素抑制方面的差异。这些观察到的差异可能是由于 REBOV VP35 IID 的稳定性增加和灵活性丧失所致，这可以通过热移位稳定性测定来证明。与此发现一致的是，REBOV VP35 IID 的 1.71-A 晶体结构表明，它与 ZEBOV VP35 IID 高度相似，整体骨架 r.m.s.d. 为 0.64 A，但在 VP35 IID 的两个亚结构域之间的连接体上包含一个额外的螺旋元件。连接体附近的突变，包括在 REBOV 和 ZEBOV 之间交换序列，表明连接体序列对可变性的容忍度有限。与以前解决的配体自由和双链 RNA 结合的 ZEBOV VP35 IID 结构一起，我们目前对 REBOV VP35 IID 的研究强调了 VP35 在免疫抑制中的重要性。在 REBOV 和 ZEBOV VP35 蛋白之间观察到的功能差异可能导致致病性观察到的差异，但这些差异不太可能是主要决定因素。然而，结构上的高度相似性和序列可变性的低容忍度，再加上埃博拉病毒 VP35 蛋白的多个关键作用，突出了 VP35 作为潜在治疗靶点的可行性。
