Department of Veterinary Medicine, College of Animal Science and Technology, Zhejiang A&F University, Hangzhou, Zhejiang Province, China.
Department of Veterinary Medicine, College of Animal Science and Technology, Zhejiang A&F University, Hangzhou, Zhejiang Province, China
J Virol. 2018 Jan 2;92(2). doi: 10.1128/JVI.01345-17. Print 2018 Jan 15.
Infectious bursal disease virus (IBDV) is a bisegmented double-strand RNA (dsRNA) virus of the family. While IBDV genomic dsRNA lacks a 5' cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts were directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by -supplementation of the viral proteins VP1 and VP3 which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5' and 3' untranslated regions (UTRs) of IBDV dsRNA were essential for VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, downregulation or inhibition of the cap-binding protein eIF4E did not decrease but, rather, enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of the 5' cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses. A key point of control for virus replication is viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery and is thus noninfectious. Our results constitute the first direct experimental evidence that VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute for cap and replacing the cap-binding protein eIF4E. Significantly, VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense strand but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strands of the viral genomic dsRNA.
传染性腔上囊病病毒（IBDV）是一种双节段双链 RNA（dsRNA）病毒，属于呼肠孤病毒科。虽然 IBDV 基因组 dsRNA 缺乏 5'帽结构，但 uncapped IBDV 基因组 RNA 有效翻译的机制尚不清楚。在本研究中，我们描述了一种依赖 VP1 和 VP3 的 IBDV uncapped RNA 翻译起始的 cap-independent 途径。我们发现，纯化的 IBDV 基因组 dsRNA 或 uncapped 病毒正链 RNA 转录本均不能直接在宿主细胞中翻译并拯救成感染性病毒。这种 uncapped IBDV 基因组 dsRNA 翻译缺陷可通过添加病毒蛋白 VP1 和 VP3 得到挽救，这依赖于 VP1 的完整聚合酶活性和 VP3 的 dsRNA 结合活性。缺失分析表明，IBDV dsRNA 的 5'和 3'非翻译区（UTR）均对 VP1/VP3 依赖性翻译起始至关重要。重要的是，VP1 和 VP3 还可以介导从 IBDV dsRNA 的真实负链恢复感染性 IBDV。此外，下调或抑制帽结合蛋白 eIF4E 不仅没有降低，反而增强了 VP1/VP3 介导的 uncapped IBDV RNA 的翻译。总之，我们的研究结果首次揭示了 VP1 和 VP3 可以弥补 5'帽的缺陷，并取代 eIF4E，赋予 uncapped IBDV RNA 被翻译并拯救成感染性病毒的能力。病毒复制的一个关键控制点是病毒翻译起始。本研究表明，uncapped IBDV RNA 不能直接被宿主翻译机制翻译为病毒蛋白，因此是非感染性的。我们的结果首次直接证明了 VP1 和 VP3 是必需的和充分的，它们可以作为帽结构的替代物并取代帽结合蛋白 eIF4E，从而启动 uncapped IBDV 基因组 RNA 的翻译。重要的是，VP1/VP3 不仅可以从正链，还可以从 IBDV dsRNA 的负链恢复感染性 IBDV。这些发现不仅为 IBDV 的生命周期分子机制提供了新的见解，也为从 IBDV 基因组 dsRNA 的正链和负链恢复 IBDV 提供了一种新的替代策略。
