Tao Wanyin, Gan Tianyu, Guo Mingzhe, Xu Yongfen, Zhong Jin
Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
University of Chinese Academy of Sciences, Beijing, China.
J Virol. 2017 Oct 27;91(22). doi: 10.1128/JVI.01316-17. Print 2017 Nov 15.
Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The minigenomic RNA harboring a luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the EBOV minigenome demonstrated that the stable EBOV replicon had much-more-active minigenome replication than previously developed transient-transfection-based EBOV minigenome systems, which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients. The scope and severity of the recent Ebola outbreak in Western Africa justified a more comprehensive investigation of the causative risk group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows experimentation under biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The replicon system had more-active genome replication than previously developed transient-transfection-based EBOV minigenome systems, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing, and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.
埃博拉病毒（EBOV）可导致人类和其他灵长类动物出现严重出血热，病死率很高。目前尚无获批的针对埃博拉病毒的药物或疫苗，因此有必要更好地了解该病毒的生命周期。由于涉及活病毒的实验仅限于生物安全4级（BSL4）实验室，埃博拉病毒的研究受到了阻碍。埃博拉病毒微型基因组系统为研究人员提供了在BSL2条件下研究埃博拉病毒的机会。在此，我们开发了一种新型埃博拉病毒微型基因组复制子，据我们所知，这是首个能够稳定复制和转录埃博拉病毒微型基因组的埃博拉病毒细胞培养系统。携带荧光素酶和潮霉素抗性标记的微型基因组RNA能够在稳定表达病毒核蛋白（NP）、病毒蛋白35（VP35）、VP30和L蛋白的辅助细胞中复制数月。对埃博拉病毒微型基因组的病毒RNA（vRNA）、互补RNA（cRNA）和信使RNA（mRNA）水平进行定量分析表明，与之前开发的基于瞬时转染的埃博拉病毒微型基因组系统相比，这种稳定的埃博拉病毒复制子具有更活跃的微型基因组复制能力，后者更多地是模拟病毒的初级转录而非基因组复制。有趣的是，稳定的埃博拉病毒复制子细胞中的微型基因组复制对干扰素治疗或RNA干扰不敏感。此外，对复制子细胞裂解物进行核糖核酸酶消化后发现，埃博拉病毒微型基因组vRNA核糖核蛋白复合体具有显著的稳定性，这可能有助于加深对埃博拉病毒在康复患者体内持续存在的理解。近期西非埃博拉疫情的范围和严重程度证明有必要对致病的4类风险病原体埃博拉病毒（EBOV）进行更全面的调查。开发一种能够在生物安全2级条件下进行实验的细胞培养系统，有助于埃博拉病毒复制及抗病毒药物研发的研究。在此，我们开发了一种新型的稳定埃博拉病毒微型基因组复制子，据我们所知，这是首个能够稳定复制和转录埃博拉病毒微型基因组的埃博拉病毒细胞培养系统。该复制子系统具有比之前开发的基于瞬时转染的埃博拉病毒微型基因组系统更活跃的基因组复制能力，为研究埃博拉病毒复制提供了一个便捷的替代系统。此外，复制子细胞中自我复制的微型基因组vRNA在干扰素治疗、RNA沉默和核糖核酸酶消化作用下表现出很强的稳定性，这可能为埃博拉病毒在幸存者体内持续存在提供了解释。
