Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66103, USA.
J Biol Chem. 2011 Apr 1;286(13):11814-24. doi: 10.1074/jbc.M110.210179. Epub 2011 Feb 4.
Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of the viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and the 5' UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.
汉坦病毒属于布尼亚病毒科，是负链 RNA 新兴病毒，也是 A 类病原体，当通过感染啮齿动物宿主的气溶胶化排泄物传播给人类时，会导致严重疾病。汉坦病毒进化出了一种新颖的翻译起始机制，由核衣壳蛋白（N）操纵，该机制优先促进病毒 mRNA 的翻译。N 与核糖体蛋白 S19（RPS19）结合，后者是 40S 核糖体亚基的结构成分。此外，N 还与病毒 mRNA 5' 帽和病毒 mRNA 5'UTR 的高度保守三重复序列结合。N 在末端帽和 5'UTR 上的同时结合有利于核糖体在翻译起始时加载到病毒转录本上。我们描述了 N 与 RPS19 之间的结合，并证明了 N-RPS19 相互作用在 N 介导的翻译起始机制中的作用。我们表明 N 与 RPS19 具有高亲和力和 1:1 的结合化学计量比特异性结合。N-RPS19 相互作用是一个焓驱动的过程。RPS19 在与 N 结合后发生构象变化。使用 T7 RNA 聚合酶，我们合成了汉坦病毒 S 片段 mRNA，其与细胞中由病毒 RNA 依赖性 RNA 聚合酶生成的转录本匹配。我们表明，N-RPS19 相互作用在细胞和兔网织红细胞裂解物中均对该 mRNA 的翻译起关键作用。我们的结果表明，N 介导的翻译起始机制，即诱使宿主翻译机制优先翻译病毒转录本，主要取决于 N-RPS19 相互作用。我们建议 N-RPS19 相互作用是一种新的靶点，可以关闭 N 介导的翻译策略，从而抑制细胞中的病毒复制。
