Millet Jean Kaoru, Whittaker Gary R
Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States.
Bio Protoc. 2016 Dec 5;6(23). doi: 10.21769/BioProtoc.2035.
Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay.
病毒假型颗粒（pp）是包膜病毒颗粒，通常源自逆转录病毒或弹状病毒，其表面带有异源包膜糖蛋白且基因组缺乏必需基因。这些合成病毒颗粒是天然病毒更安全的替代物，并具有由所使用的异源包膜糖蛋白决定的嗜性和宿主进入途径特征。它们已被证明是非常有用的研究工具，有许多应用，例如用于研究包膜病毒的进入途径以及生成有效的基因递送载体。其产生的基础在于某些病毒，如鼠白血病病毒（MLV），能够将其他病毒的包膜糖蛋白整合到假型病毒颗粒中。这些颗粒可以设计成包含荧光素酶等报告基因，从而在假型颗粒感染易感细胞时能够对病毒进入事件进行定量。在此，我们详细介绍一种以中东呼吸综合征冠状病毒（MERS-CoV）刺突蛋白（S）作为要整合的异源包膜糖蛋白为例，生成基于MLV的假型颗粒的方案。我们还描述了如何使用这些颗粒感染易感细胞，并通过荧光素酶测定进行定量感染性读数。
