Department of Chemistry, University of Manitoba, Parker Building, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2.
Department of Chemistry, University of Manitoba, Parker Building, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; Manitoba Group in Protein Structure and Function, University of Manitoba, Parker Building, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2.
J Mol Biol. 2012 Oct 5;422(5):635-649. doi: 10.1016/j.jmb.2012.06.017. Epub 2012 Jun 16.
Foreign double-stranded RNA (dsRNA) generated during the normal course of the viral life cycle serves as a key infection recognition element by proteins of the innate immune response. To circumvent this response, all adenoviruses synthesize at least one highly structured RNA (VA(I)), which, after processing by the RNA silencing machinery, inhibits the innate immune response via a series of interactions with specific protein partners. Surprisingly, VA(I) positively regulates the activity of the interferon-induced 2'-5'-oligoadenylate synthetase (OAS) enzymes, which typically represent a key mechanism whereby host-cell protein translation is attenuated in response to foreign dsRNA. We present data investigating the regulation of the OAS1 isoform by VA(I) derivatives and demonstrate that a processed version of VA(I) lacking the terminal stem behaves as a pseudo-inhibitor of OAS1. A combination of electrophoretic mobility shift assays, dynamic light scattering, and non-denaturing mass spectrometry was used to quantitate binding affinity and characterize OAS1:VA(I) complex stoichiometry. Enzyme assays characterized the ability of VA(I) derivatives to activate OAS1. Finally, the importance of RNA 5'-end phosphorylation state is investigated, and it emphasizes its potential importance in the activation or inhibition of OAS enzymes. Taken together, these data suggest a plausible strategy whereby the virus produces a single RNA transcript capable of inhibiting a variety of members of the innate immune response.
在病毒生命周期的正常过程中产生的外来双链 RNA (dsRNA) 作为先天免疫反应蛋白的关键感染识别元件。为了规避这种反应，所有腺病毒至少合成一种高度结构化的 RNA (VA[I])，后者在 RNA 沉默机制加工后，通过与特定蛋白伴侣的一系列相互作用来抑制先天免疫反应。令人惊讶的是，VA[I] 正向调节干扰素诱导的 2'-5'-寡腺苷酸合成酶 (OAS) 酶的活性，该酶通常是宿主细胞蛋白翻译在响应外来 dsRNA 时被减弱的关键机制。我们提供了有关 VA[I] 衍生物对 OAS1 调节的数据，并证明缺乏末端茎的 VA[I] 的处理版本表现为 OAS1 的伪抑制剂。使用电泳迁移率变动分析、动态光散射和非变性质谱联用技术来定量结合亲和力并表征 OAS1：VA(I) 复合物的化学计量比。酶分析表征了 VA[I] 衍生物激活 OAS1 的能力。最后，研究了 RNA 5'-端磷酸化状态的重要性，并强调了其在 OAS 酶的激活或抑制中的潜在重要性。总之，这些数据表明病毒产生一种单一的 RNA 转录本，能够抑制先天免疫反应的多种成员。
