Department of Biochemistry and Molecular Biology, University of British Columbiagrid.17091.3e, Vancouver, Canada.
Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbiagrid.17091.3e, Vancouver, Canada.
J Virol. 2022 Mar 9;96(5):e0133021. doi: 10.1128/JVI.01330-21. Epub 2022 Jan 12.
All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an intergenic region internal ribosome entry site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that comprises a ribosome binding domain of pseudoknot II and III (PKII and PKIII), and a tRNA-like anticodon domain (PKI) connected via a short, one to three nucleotide hinge region. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In this study, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation . Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. Viruses must use the host cell machinery to direct viral protein expression for productive infection. One such mechanism is an internal ribosome entry site that can directly recruit host cell machinery. In this study, we have identified a novel sequence in an IRES that provides insight into the mechanism of viral gene expression. Specifically, this novel sequence promotes viral IRES activity by directly guiding the host cell machinery to start gene expression at a specific site.
所有病毒都必须盗用宿主核糖体来进行病毒蛋白合成。双顺反子病毒利用基因间内部核糖体进入位点（IGR IRES）直接招募核糖体，并介导从非 AUG 起始密码子开始翻译起始。IGR IRES 采用三假结结构，包括假结 II 和假结 III（PKII 和 PKIII）的核糖体结合结构域，以及通过短的一到三个核苷酸铰链区连接的 tRNA 样反密码子结构域（PKI）。最近对双顺反子病毒 Taura 综合征病毒（TSV）IGR IRES 与核糖体结合的冷冻电镜结构分析表明，铰链区可能有助于 IRES 从核糖体 A 位到 P 位的易位。在这项研究中，我们提供了关于铰链区在 IGR IRES 翻译中的作用的机制和功能见解。使用蜜蜂双顺反子病毒以色列急性麻痹病毒（IAPV）作为模型，我们证明了铰链区突变导致 IRES 依赖性翻译减少。对突变 IRES 结合到纯化核糖体和兔网织红细胞裂解物的 toe-printing 引物延伸分析显示，在 IRES 上初始核糖体定位存在缺陷。最后，使用杂种双顺反子克隆，IAPV IRES 铰链区的突变导致病毒产量降低。我们的工作揭示了双顺反子病毒 IGR IRES 铰链区的一个意外作用，它协调 IRES 的两个独立折叠结构域，使核糖体正确定位以开始翻译。病毒必须利用宿主细胞机制来指导病毒蛋白表达以进行有效感染。一种这样的机制是内部核糖体进入位点，它可以直接招募宿主细胞机制。在这项研究中，我们已经确定了 IRES 中的一个新序列，该序列提供了对病毒基因表达机制的深入了解。具体来说，这个新序列通过直接指导宿主细胞机制在特定位置开始基因表达，从而促进病毒 IRES 活性。
