Profiling of rotavirus 3UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication
Profiling of rotavirus 3UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication
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DOI：
10.1074/jbc.ra118.006004
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发表时间：
2019-04-12
影响因子：
4.8
通讯作者：
Greenberg, Harry B.
中科院分区：
文献类型：
--
作者：
Ren, Lili;Ding, Siyuan;Greenberg, Harry B.
节段RNA病毒的基因组复制和病毒体组装是高度协调的事件，受到病毒RNA UTR中的序列和结构元件的严格调控。该过程定义不明确，可能需要宿主蛋白与病毒蛋白协同参与。在这项研究中，我们采用了一种基于蛋白质组学的方法，命名为RNA-蛋白质相互作用检测（RaPID），全面筛选结合到轮状病毒（RV）3末端保守基序的宿主蛋白。使用该测定，我们鉴定了ATP 5 B，线粒体ATP合酶的核心亚基，其对RV 3UTR共有序列具有高亲和力。在RV感染过程中，ATP 5 B与RV 3UTR结合，并与病毒RNA和病毒质共定位。在功能上，siRNA介导的ATP 5 B或其他ATP合酶亚基（例如ATP 5A 1和ATP 5 O）的基因缺失减少了感染性病毒后代的产生，而细胞内病毒RNA水平或RNA翻译没有显着改变。ATP合成酶的化学抑制减少RV产量在传统的细胞培养和人类肠上皮细胞，表明ATP 5 B正调控晚期RV成熟的原代肠上皮细胞。总的来说，我们的研究结果揭示了宿主蛋白在RV基因组组装和颗粒形成中的作用，并将ATP 5 B鉴定为一种新的前RV RNA结合蛋白，有助于我们理解宿主ATP酶如何刺激病毒生长和发病机制。
Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA-protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3 terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3UTR consensus sequences. During RV infection, ATP5B bound to the RV 3UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.