King Benjamin R, Hershkowitz Dylan, Eisenhauer Philip L, Weir Marion E, Ziegler Christopher M, Russo Joanne, Bruce Emily A, Ballif Bryan A, Botten Jason
Department of Medicine, Division of Immunobiology, University of Vermont, Burlington, Vermont, USA.
Cellular, Molecular, and Biomedical Sciences Graduate Program, University of Vermont, Burlington, Vermont, USA.
J Virol. 2017 Jul 12;91(15). doi: 10.1128/JVI.00763-17. Print 2017 Aug 1.
Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV. Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.
沙粒病毒是有包膜的负链RNA病毒，可导致严重的人类疾病。这些病毒仅编码四种蛋白质来完成病毒生命周期，因此每种沙粒病毒蛋白在感染过程中可能都发挥着尚未被认识的辅助作用。在这里，我们使用免疫沉淀和质谱法来鉴定与旧大陆沙粒病毒淋巴细胞性脉络丛脑膜炎病毒（LCMV）和新大陆沙粒病毒胡宁病毒（JUNV）毒株Candid #1的核蛋白（NP）相互作用的人类蛋白质。对NP已鉴定的蛋白质伙伴进行生物信息学分析表明，宿主翻译似乎是感染过程中涉及的关键生物学过程。特别是，NP与双链RNA（dsRNA）激活的蛋白激酶（PKR）相关联，PKR是一种特征明确的抗病毒蛋白，它通过磷酸化eIF2α来抑制帽依赖性蛋白翻译起始。JUNV感染导致PKR表达增加及其重新分布到病毒复制和转录工厂。此外，PKR的磷酸化是其磷酸化eIF2α能力的先决条件，很容易被JUNV诱导。然而，即使在暴露于合成双链RNA聚肌苷酸胞嘧啶核苷酸（poly(I·C)）（一种有效的PKR激动剂）后，JUNV也能阻止这部分活化的PKR磷酸化eIF2α。PKR功能的这种阻断具有高度特异性，因为LCMV无法同样抑制eIF2α磷酸化。JUNV拮抗PKR抗病毒活性的能力似乎是完全的，因为沉默PKR表达对病毒繁殖没有影响。总之，我们提供了沙粒病毒NP所涉及的宿主机制的详细图谱，并确定了一条被JUNV颠覆的抗病毒途径。沙粒病毒是重要的人类病原体，目前尚无FDA批准的疫苗，需要有效的抗病毒治疗方法。抗病毒治疗方案的设计和疾病发病机制的阐明将取决于对这些病毒，特别是它们与宿主细胞机制相互作用的更深入基础理解。鉴定对病毒生命周期和/或发病机制至关重要的宿主蛋白是发现新药物靶点的有用策略。这项研究首次揭示了沙粒病毒核蛋白广泛的人类蛋白质相互作用组，并发现了一种在胡宁病毒感染期间被中和的强大抗病毒宿主蛋白。这样做进一步深入了解了病毒与宿主先天免疫反应之间的相互作用，并为该领域提供了重要的数据集。
