Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth
Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth
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DOI：
10.1128/jvi.01144-12
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发表时间：
2012-10-01
影响因子：
5.4
通讯作者：
Albarino, Cesar G.
中科院分区：
文献类型：
--
作者：
Bergeron, Eric;Chakrabarti, Ayan K.;Albarino, Cesar G.
沙粒病毒是啮齿动物传播的病毒，具有双片段RNA基因组。最近在赞比亚和南非发现了一种遗传上独特的沙粒病毒Lujo病毒，它是院内爆发的有出血性表现的急性发热性疾病的致病因子。这次爆发的病死率为80%。建立了从cDNA中拯救传染性Lujo病毒的反向遗传系统，研究了该病毒的生物学特性。基因组末端测序显示在S段启动子元件的3'端有独特的核苷酸。在开发该系统的过程中，我们发现利用先前报道的含有沙粒病毒转录终止信号的L段基因间区（IGR）重建传染性Lujo病毒，产生了减毒的Lujo病毒。重测序显示，正确的L段IGR长36个核苷酸，将其纳入重建的Lujo病毒中，使其生长速度恢复到真实的临床病毒分离物的生长速度。预计这些额外的核苷酸将使IGR主茎环结构的自由能增加一倍以上。此外，将新确定的L- igr纳入复制子报告系统可增强荧光素酶报告基因L片段的表达。总之，这些结果表明，L-IGR中一个极其稳定的二级结构对Lujo病毒的繁殖和病毒蛋白的产生至关重要。生产重组Lujo病毒的技术现在为精确研究这种新发现的病原体毒力的分子决定因素提供了一种方法。
Arenaviruses are rodent-borne viruses with a bisegmented RNA genome. A genetically unique arenavirus, Lujo virus, was recently discovered as the causal agent of a nosocomial outbreak of acute febrile illness with hemorrhagic manifestations in Zambia and South Africa. The outbreak had a case fatality rate of 80%. A reverse genetics system to rescue infectious Lujo virus from cDNA was established to investigate the biological properties of this virus. Sequencing the genomic termini showed unique nucleotides at the 3' terminus of the S segment promoter element. While developing this system, we discovered that reconstructing infectious Lujo virus using the previously reported L segment intergenic region (IGR), comprising the arenaviral transcription termination signal, yielded an attenuated Lujo virus. Resequencing revealed that the correct L segment IGR was 36 nucleotides longer, and incorporating it into the reconstructed Lujo virus restored the growth rate to that of the authentic clinical virus isolate. These additional nucleotides were predicted to more than double the free energy of the IGR main stem-loop structure. In addition, incorporating the newly determined L-IGR into a replicon reporter system enhanced the expression of a luciferase reporter L segment. Overall, these results imply that an extremely stable secondary structure within the L-IGR is critical for Lujo virus propagation and viral protein production. The technology for producing recombinant Lujo virus now provides a method to precisely investigate the molecular determinants of virulence of this newly identified pathogen.