Furuyama Wakako, Sakaguchi Miako, Yamada Kento, Nanbo Asuka
Department of Virus Infection Dynamics, National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki, Japan.
Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan.
Front Microbiol. 2022 Nov 3;13:1026644. doi: 10.3389/fmicb.2022.1026644. eCollection 2022.
Ebola virus (EBOV) causes severe EBOV disease (EVD) in humans and non-human primates. Currently, limited countermeasures are available, and the virus must be studied in biosafety level-4 (BSL-4) laboratories. EBOV glycoprotein (GP) is a single transmembrane protein responsible for entry into host cells and is the target of multiple approved drugs. However, the molecular mechanisms underlying the intracellular dynamics of GP during EBOV lifecycle are poorly understood. In this study, we developed a novel GP monitoring system using transcription- and replication-competent virus-like particles (trVLPs) that enables the modeling of the EBOV lifecycle under BSL-2 conditions. We constructed plasmids to generate trVLPs containing the coding sequence of EBOV GP, in which the mucin-like domain (MLD) was replaced with fluorescent proteins. The generated trVLP efficiently replicated over multiple generations was similar to the wild type trVLP. Furthermore, we confirmed that the novel trVLP system enabled real-time visualization of GP throughout the trVLP replication cycle and exhibited intracellular localization similar to that of wild type GP. In summary, this novel monitoring system for GP will enable the characterization of the molecular mechanism of the EBOV lifecycle and can be applied for the development of therapeutics against EVD.
埃博拉病毒（EBOV）可导致人类和非人类灵长类动物患上严重的埃博拉病毒病（EVD）。目前，可用的应对措施有限，且该病毒必须在生物安全4级（BSL-4）实验室中进行研究。埃博拉病毒糖蛋白（GP）是一种负责进入宿主细胞的单跨膜蛋白，也是多种已获批药物的靶点。然而，在埃博拉病毒生命周期中，GP细胞内动态变化的分子机制仍知之甚少。在本研究中，我们开发了一种新型的GP监测系统，该系统使用具有转录和复制能力的病毒样颗粒（trVLPs），能够在BSL-2条件下模拟埃博拉病毒的生命周期。我们构建了质粒以产生包含埃博拉病毒GP编码序列的trVLPs，其中粘蛋白样结构域（MLD）被荧光蛋白取代。所产生的trVLP在多代中高效复制，与野生型trVLP相似。此外，我们证实这种新型trVLP系统能够在整个trVLP复制周期实时可视化GP，并表现出与野生型GP相似的细胞内定位。总之，这种新型的GP监测系统将有助于阐明埃博拉病毒生命周期的分子机制，并可应用于埃博拉病毒病治疗药物的开发。
