Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.
Biochemistry. 2013 Nov 26;52(47):8406-19. doi: 10.1021/bi400704d. Epub 2013 Nov 14.
Viral protein 35 (VP35), encoded by filoviruses, is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion, and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for nonenzymatic targets, are often challenging. Our previous work on filoviral VP35 proteins defined conserved basic residues located within its C-terminal dsRNA binding interferon (IFN) inhibitory domain (IID) as important for VP35 mediated IFN antagonism and viral polymerase cofactor functions. In the current study, we used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target for aptamer selection using SELEX. Select aptamers, representing, two distinct classes, were further characterized based on their interaction properties to eVP35 IID. These results revealed that these aptamers bind to distinct regions of eVP35 IID with high affinity (10-50 nM) and specificity. These aptamers can compete with dsRNA for binding to eVP35 and disrupt the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize the eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by the expression of select viral proteins. Taken together, our results support the identification of two aptamers that bind filoviral VP35 proteins with high affinity and specificity and have the capacity to potentially function as filoviral VP35 protein inhibitors.
病毒蛋白 35（VP35），由丝状病毒编码，是一种多功能的 dsRNA 结合蛋白，在病毒复制、先天免疫逃避和发病机制中发挥重要作用。这些蛋白质的多功能性质也为开发针对不同功能区域的对策提供了机会。然而，针对这些多功能蛋白质（特别是非酶靶标）进行功能验证和建立治疗方法通常具有挑战性。我们之前关于丝状病毒 VP35 蛋白的工作确定了位于其 C 端 dsRNA 结合干扰素（IFN）抑制域（IID）内的保守碱性残基对 VP35 介导的 IFN 拮抗和病毒聚合酶辅助因子功能很重要。在当前的研究中，我们使用结构和功能数据的组合来确定埃博拉病毒（EBOV）VP35（eVP35）的区域，以使用 SELEX 进行适体选择。选择的适体代表两个不同的类别，进一步根据它们与 eVP35 IID 的相互作用特性进行了表征。这些结果表明，这些适体以高亲和力（10-50 nM）和特异性结合到 eVP35 IID 的不同区域。这些适体可以与 dsRNA 竞争结合 eVP35 并破坏 eVP35-核蛋白（NP）相互作用。与拮抗 eVP35-NP 相互作用的能力一致，选择的适体可以抑制通过表达选择的病毒蛋白重新构成的 EBOV 聚合酶复合物的功能。总之，我们的结果支持鉴定两种与丝状病毒 VP35 蛋白具有高亲和力和特异性的适体，并且有可能作为丝状病毒 VP35 蛋白抑制剂发挥作用。
