Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA; Biochemistry Undergraduate Program, Iowa State University, Ames, IA 50011, USA.
Simulation Sciences Branch, US Army Research Laboratory, Aberdeen, MD 21005, USA; Department of Cell Biology and Biochemistry, USAMRIID, 1425 Porter St., Fort Detrick, MD 21702, USA.
J Mol Biol. 2014 May 15;426(10):2045-58. doi: 10.1016/j.jmb.2014.01.010. Epub 2014 Feb 1.
The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
埃博拉病毒（EBOV）基因组仅编码一种具有酶活性的单一病毒多肽，即病毒的大（L）RNA 依赖性 RNA 聚合酶蛋白。然而，目前关于 L 蛋白的信息有限，这阻碍了抗病毒药物的开发。因此，抗丝状病毒的治疗努力必须包括其他靶点，如蛋白质-蛋白质界面。病毒蛋白 35（VP35）具有多功能性，在病毒发病机制中发挥重要作用，包括病毒 mRNA 合成和负义 RNA 病毒基因组的复制。先前的研究表明，VP35 干扰素抑制结构域（IID）内关键碱性残基的突变会导致 EBOV 在体外和体内显著衰减。在本研究中，我们使用了一种实验管道，包括基于结构的计算筛选以及生化和结构表征，以及药物化学，以鉴定和表征针对 VP35 结合口袋的小分子。NMR 映射实验和高分辨率 X 射线晶体结构表明，选择的小分子结合到 VP35 IID 的一个区域，该区域通过与病毒核蛋白（NP）的相互作用对于复制复合物的形成很重要。我们还测试了选择的化合物抑制 VP35 IID-NP 相互作用的能力，以及在 minigenome 测定和 EBOV 复制中 VP35 的功能。这些结果证实了本研究中鉴定的化合物能够抑制 VP35-NP 相互作用以及在基于细胞的测定中损害病毒复制的能力。这些研究为针对丝状病毒 VP35 蛋白开发抗丝状病毒化合物提供了初步框架。
