Development of a Single-Cycle Infectious SARS-CoV-2 Virus Replicon Particle System for Use in Biosafety Level 2 Laboratories.
Development of a Single-Cycle Infectious SARS-CoV-2 Virus Replicon Particle System for Use in Biosafety Level 2 Laboratories.
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DOI：
10.1128/jvi.01837-21
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发表时间：
2022-02-09
影响因子：
5.4
通讯作者：
Manicassamy B
中科院分区：
文献类型：
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作者：
Malicoat J;Manivasagam S;Zuñiga S;Sola I;McCabe D;Rong L;Perlman S;Enjuanes L;Manicassamy B
传染性严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的研究活动目前只有在生物安全3级(BSL3)控制下才被允许。在这里，我们报道了一个单周期传染性SARS-CoV-2病毒复制子颗粒(VRP)系统的发展，该系统带有荧光素酶和绿色荧光蛋白(GFP)双重报告，可以在BSL2实验室安全地操作，以研究SARS-CoV-2生物学。SARS-CoV-2的S蛋白(S)编码包膜糖蛋白，它是介导新宿主细胞感染所必需的。通过缺失和用荧光素酶和绿色荧光蛋白双重报告基因替换这个必需的S基因，我们产生了一个条件突变株(ΔS-vrp)，它只在表达病毒包膜糖蛋白的细胞中产生感染性颗粒。有趣的是，我们观察到表达水泡性口炎病毒糖蛋白G[ΔS-vrp(G)]的细胞比表达包括S在内的其他病毒糖蛋白的细胞更有效地产生感染性颗粒。我们证实，ΔS-vrp(G)的感染仅限于一轮，并可被抗水泡性口炎病毒血清中和。在我们对ΔS-VRP(G)的研究中，我们观察到荧光素酶和绿色荧光蛋白在不同类型的人和小鼠细胞中都有强劲的表达，证明了多种细胞可以支持SARS-CoV-2的细胞内复制。此外，用抗冠状病毒药物雷米地韦(核苷类似物)和GC376(冠状病毒3CL蛋白酶抑制剂)处理ΔS-维拉帕米(G)感染的细胞后，荧光素酶和绿色荧光蛋白的表达均显著降低，并呈药物剂量和细胞类型的依赖关系。综上所述，我们的研究结果表明，我们已经开发出一个单周期传染性SARS-CoV-2 VRP系统，作为一个通用的平台，用于研究SARS-CoV-2的细胞内生物学和在BSL2包涵体下进行抗病毒药物的高通量筛选。由于SARS-CoV-2的高度传染性和人类缺乏免疫力，对SARS-CoV-2的研究仅限于生物安全三级实验室。这极大地限制了更广泛的科学界对SARS-CoV-2研究的参与，从而阻碍了疫苗和抗病毒药物的开发。通过删除病毒基因组中的关键刺突基因，我们开发了一种带有荧光素酶和荧光报告分子的SARS-CoV-2条件突变体，可以在生物安全二级条件下安全使用。我们的单周期传染性SARS-CoV-2病毒复制子系统可以作为一个通用的平台来研究SARS-CoV-2的细胞内生物学和在BSL2包涵体下进行抗病毒药物的高通量筛选。
Research activities with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The spike (S) gene of SARS-CoV-2 encodes the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G [ΔS-VRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug dose- and cell-type-dependent manner. Taken together, our findings show that we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment. IMPORTANCE Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and antiviral drugs. By deleting the essential spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment.