Abstract
ABSTRACTResearch 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.
Highlights
(SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment
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
To safely study SARS-CoV-2 biology under BSL2 containment, we deleted and replaced the essential viral spike (S) ORF with a tandem Gaussia luciferase and Neon Green GFP dual reporter separated by the porcine teschovirus 2A ribosome skipping signal under the control of the S gene transcription regulatory sequence ( S-Luc-GFP; Fig 1A)
Summary
To safely study SARS-CoV-2 biology under BSL2 containment, we deleted and replaced the essential viral spike (S) ORF with a tandem Gaussia luciferase and Neon Green GFP dual reporter separated by the porcine teschovirus 2A ribosome skipping signal under the control of the S gene transcription regulatory sequence ( S-Luc-GFP; Fig 1A). As S-VRP(G) infection of host cells is independent of SARS-CoV-2 host receptor ACE2 expression, we evaluated the robustness of the S-VRP(G) dual reporter system in various cell types of human and murine origin (Fig 3A-D). We observed robust induction of various antiviral genes including Ifn , Ifn , Mx1, Isg and Tnf in BMDMs and to a lesser extent in BMDCs (Fig 3E) Taken together, these results demonstrate that the S-VRP(G) dual reporter system is a versatile tool to study SARS-CoV-2 biology as well as host immune responses in various cell types. Huh7.5 cells, indicating cell type specific differences in the activity of antiviral drugs These results demonstrate that the S-VRP(G) dual reporter system can be useful for rapid screening of antiviral drugs against SARS-CoV-2 under BSL2 containment. We have successfully established a SARS-CoV-2 VRP dual reporter platform that allows for safe investigation of SARS-CoV-2 biology, host interactions, and antiviral responses, as well as for high throughput screening of anti-CoV drugs, under BSL2 containment
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