Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
Sci Adv. 2020 Apr 15;6(16):eaay6410. doi: 10.1126/sciadv.aay6410. eCollection 2020 Apr.
In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo-electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.
在许多病毒中，包括轮状病毒（RV），它是婴儿肠胃炎的主要病原体，病毒信使 RNA 的加帽是病毒基因组高效翻译的关键步骤。在 RV 中，VP3 在颗粒核心内由 RV 聚合酶 VP1 从基因组 dsRNA 片段合成的新生转录本上进行加帽。在这里，通过低温电子显微镜、X 射线晶体学和生化分析，我们表明 VP3 形成了一个稳定的四聚体组装，每个亚基都具有模块化的结构域组织，该组织独特地整合了加帽转录本所需的五个不同的酶促步骤。除了先前已知的鸟苷酰转移酶和甲基转移酶活性外，我们还表明 VP3 具有迄今为止尚未被怀疑的 RNA 三磷酸酶活性，该酶对于起始转录本加帽是必需的，并且可能需要 RNA 解旋酶活性来分离在转录本内形成的短暂 RNA 双链。根据我们的研究，我们提出了一种新的机制，说明病毒衣壳内的 VP3 如何对从聚合酶中伸出的新生转录本进行加帽。
