Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting
Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting
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DOI：
10.3791/59010
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发表时间：
2019-03-01
影响因子：
1.2
通讯作者：
Whittaker, Gary R.
中科院分区：
文献类型：
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作者：
Millet, Jean K.;Tang, Tiffany;Whittaker, Gary R.
该方案旨在使用广泛可用的HEK-293 T细胞系的简单转染程序，产生具有鼠白血病病毒（MLV）核心和荧光素酶报告基因的冠状病毒（CoV）刺突（S）融合蛋白假型颗粒。一旦形成并从生产细胞中释放，这些假病毒体就掺入荧光素酶报告基因。由于它们在其表面上仅含有异源冠状病毒刺突蛋白，因此颗粒在进入步骤中表现得像它们的天然冠状病毒对应物。因此，它们是研究病毒进入宿主细胞的天然病毒体的优秀替代物。在成功进入和感染靶细胞后，荧光素酶报告基因整合到宿主细胞基因组中并表达。使用简单的荧光素酶测定，可以容易地定量转导的细胞。该程序的一个重要优点是，它可以在生物安全2级（BSL-2）设施中进行，而不是在处理中东呼吸综合征冠状病毒（MERS-CoV）和严重急性呼吸综合征冠状病毒（SARS-CoV）等高致病性冠状病毒所需的BSL-3设施中进行。另一个益处来自其多功能性，因为其可以应用于属于所有三类病毒融合蛋白的包膜蛋白，例如I类流感血凝素（HA）和埃博拉病毒糖蛋白（GP）、II类塞姆利基森林病毒E1蛋白或III类水泡性口炎病毒G糖蛋白。该方法的一个局限性是，它只能重演病毒的进入步骤所研究的包膜蛋白介导的。为了研究其他病毒生命周期步骤，需要其他方法。这些假型颗粒可用于的许多应用的实例包括宿主细胞易感性和向性的研究以及测试病毒进入抑制剂的作用以剖析所使用的病毒进入途径。
The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.