Thakur Nazia, Gallo Giulia, Elreafey Ahmed M E, Bailey Dalan
Viral Glycoproteins Group, The Pirbright Institute, Pirbright, Woking, UK.
The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Bio Protoc. 2021 Nov 5;11(21):e4249. doi: 10.21769/BioProtoc.4249.
For enveloped viruses, such as SARS-CoV-2, transmission relies on the binding of viral glycoproteins to cellular receptors. Conventionally, this process is recapitulated in the lab by infection of cells with isolated live virus. However, such studies can be restricted due to the availability of high quantities of replication-competent virus, biosafety precautions and associated trained staff. Here, we present a protocol based on pseudotyping to produce recombinant replication-defective lentiviruses bearing the SARS-CoV or SARS-CoV-2 attachment Spike glycoprotein, allowing the investigation of viral entry in a lower-containment facility. Pseudoparticles are produced by cells transiently transfected with plasmids encoding retroviral RNA packaging signals and proteins, for the reconstitution of lentiviral particles, and a plasmid coding for the viral attachment protein of interest. This approach allows the investigation of different aspects of viral entry, such as the identification of receptor tropism, the prediction of virus host range, and zoonotic transmission potential, as well as the characterisation of antibodies (sera or monoclonal antibodies) and pharmacological inhibitors that can block entry. Graphic abstract: SARS-CoV and SARS-CoV-2 pseudoparticle generation and applications.
对于包膜病毒，如严重急性呼吸综合征冠状病毒2（SARS-CoV-2），其传播依赖于病毒糖蛋白与细胞受体的结合。传统上，这一过程在实验室中通过用分离的活病毒感染细胞来重现。然而，由于难以获得大量具有复制能力的病毒、生物安全预防措施以及相关的专业技术人员，此类研究可能会受到限制。在此，我们提出了一种基于假型化的方案，用于生产携带SARS-CoV或SARS-CoV-2附着刺突糖蛋白的重组复制缺陷型慢病毒，从而能够在较低级别的生物安全设施中研究病毒进入细胞的过程。假病毒颗粒由瞬时转染了编码逆转录病毒RNA包装信号和蛋白质以重建慢病毒颗粒的质粒，以及编码感兴趣的病毒附着蛋白的质粒的细胞产生。这种方法能够研究病毒进入细胞过程的不同方面，例如识别受体嗜性、预测病毒宿主范围和人畜共患病传播潜力，以及鉴定能够阻断病毒进入的抗体（血清或单克隆抗体）和药理抑制剂。图形摘要：SARS-CoV和SARS-CoV-2假病毒颗粒的产生及应用。
