Assessment of Life Cycle Modeling Systems as Prediction Tools for a Possible Attenuation of Recombinant Ebola Viruses.
Assessment of Life Cycle Modeling Systems as Prediction Tools for a Possible Attenuation of Recombinant Ebola Viruses.
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DOI：
10.3390/v14051044
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发表时间：
2022-05-13
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--
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埃博拉病毒（EBOV）引起人类出血热，病死率高。在过去，许多重组ebov通过额外的转录单位或融合蛋白表达不同的报告蛋白。这些病毒是研究EBOV的重要工具，它们的用途包括高通量筛选方法，病毒蛋白的细胞间定位分析和病毒的组织分布，以及体内发病机制的研究。然而，至少在体内，与野生型病毒相比，它们都表现出衰减，而这种衰减的基础尚不清楚。不幸的是，拯救这些病毒是一个漫长且并不总是成功的过程，而且与它们合作仅限于生物安全水平（BSL） 4级实验室，因此寻找非减毒的表达报告病毒的ebov仍然具有挑战性。然而，已经开发了几个生命周期建模系统来模拟BSL-1或-2条件下线状病毒生命周期的不同方面，但尚不清楚这些系统是否可以用于预测重组ebov的生存能力和可能的衰减。为了解决这个问题，我们系统地将N-或c -末端的flag-HA标签或绿色荧光蛋白（GFP）融合到不同的EBOV蛋白上，并分析了这些添加对生命周期建模系统中蛋白质功能的影响。基于这些结果，选择了编码这些标签/蛋白质的重组ebov，并对其进行了体外可能衰减的表征，并将结果与生命周期建模系统的数据进行了比较。虽然小分子标记的结果显示大部分一致性良好，但基于生命周期建模系统的结果，表达gfp的病毒比预期的更弱，这表明了这些系统的局限性，并强调了与感染性病毒合作的重要性。尽管如此，生命周期建模系统仍然是排除不可行的标记策略的有用工具。
Ebola virus (EBOV) causes hemorrhagic fever in humans with high case fatality rates. In the past, a number of recombinant EBOVs expressing different reporters from additional transcription units or as fusion proteins have been rescued. These viruses are important tools for the study of EBOV, and their uses include high throughput screening approaches, the analysis of intercellular localization of viral proteins and of tissue distribution of viruses, and the study of pathogenesis in vivo. However, they all show, at least in vivo, attenuation compared to wild type virus, and the basis of this attenuation is only poorly understood. Unfortunately, rescue of these viruses is a lengthy and not always successful process, and working with them is restricted to biosafety level (BSL)-4 laboratories, so that the search for non-attenuated reporter-expressing EBOVs remains challenging. However, several life cycle modeling systems have been developed to mimic different aspects of the filovirus life cycle under BSL-1 or -2 conditions, but it remains unclear whether these systems can be used to predict the viability and possible attenuation of recombinant EBOVs. To address this question, we systematically fused N- or C-terminally either a flag-HA tag or a green fluorescent protein (GFP) to different EBOV proteins, and analyzed the impact of these additions with respect to protein function in life cycle modeling systems. Based on these results, selected recombinant EBOVs encoding these tags/proteins were then rescued and characterized for a possible attenuation in vitro, and results compared with data from the life cycle modeling systems. While the results for the small molecular tags showed mostly good concordance, GFP-expressing viruses were more attenuated than expected based on the results from the life cycle modeling system, demonstrating a limitation of these systems and emphasizing the importance of work with infectious virus. Nevertheless, life cycle modeling system remain useful tools to exclude non-viable tagging strategies.
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