Development of a Single-Cycle Infectious SARS-CoV-2 Virus Replicon Particle System for Use in Biosafety Level 2 Laboratories

Abstract

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.

Keywords: SARS-CoV-2; replicon.

FIG 1

Development of single-cycle infectious SARS-CoV-2 replicon system with a dual reporter. (A) Schematic representation of SARS-CoV-2 and ΔS Luc-GFP SARS-CoV-2 genomes. The S gene in the SARS-CoV-2 genome was replaced with a luciferase and GFP dual reporter. (B) Generation and amplification of ΔS virus replicon particles. (Left) 293T/Huh7.5 cell mixture was transfected with ΔS Luc-GFP bacmid and VSV-G plasmid. (Right) Supernatants from ΔS-VRP(G) rescue transfections were amplified in Huh7.5 cells transfected with VSV-G plasmid. (C and D) Kinetics of luciferase and GFP expression during the rescue transfection process. 293T/Huh7.5 cells were cotransfected with ΔS Luc-GFP bacmid and VSV-G plasmid, and at various days posttransfection, luciferase activity in the supernatants and GFP expression in the cell mixture were assessed. On day 6 posttransfection, 293T/Huh7.5 cells were again transfected with additional VSV-G plasmid. Luciferase values (C) and GFP expression (D) are shown at the indicated time points. Luciferase activity is shown for 3 independent Bac clones. GFP expression is shown for Bac clone 7. (E) Immunofluorescence imagining of GFP and nucleoprotein expression in ΔS-VRP(G)-infected cells. A549-hACE2 cells were infected with ΔS-VRP(G), and at 18 hpi, cells were stained with anti-N antibody and imaged. (F) Western blot analysis of nucleoprotein expression in ΔS-VRP(G)-infected cells. A549-hACE2 cells were infected with ΔS-VRP(G), and at 18 hpi, cell lysates were collected and analyzed for nucleoprotein and spike protein expression by Western blotting. Cell lysates from wild-type SARS-CoV-2-infected cells were included as controls. β-Actin levels are shown as loading controls.

FIG 2

Characterization of ΔS-VRP dual reporter system. (A) ΔS-VRP(G) infection is restricted to a single round. Huh7.5 cells were infected with ΔS-VRP(G), and at 2 hpi, cells were washed and incubated in fresh medium. At 48 h, supernatants (round 2 [R2 sup]) were collected and added to new Huh7.5 cells. At 18 hpi, luciferase activity in the supernatant was measured. (B) Comparison of infectivity of different ΔS-VRP(G) preparations. Huh7.5 cells were infected with 3 independent preparations of ΔS-VRP(G), and luciferase activity was measured at 18 hpi. (C) Infectivity of ΔS-VRP(G) stored at −80°C. Huh7.5 cells were infected with ΔS-VRP(G) stored at −80°C or fresh preparations (FS), and luciferase activity was measured at 18 hpi. (D and E) Neutralization of ΔS-VRP(G) infection by anti-VSV sera. Sera from control (Cntrl) and VSV-infected mice were preincubated with ΔS-VRP(G) for 1 h and subsequently incubated with Huh7.5 cells for 2 h. Luciferase and GFP expression were assessed at 18 hpi. For panels A to C, luciferase activity in the supernatants was measured and is shown as relative light units (RLU). For panel E, luciferase activity was normalized to the no-treatment control and is shown as a percentage of the no-treatment control.

FIG 3

Permissiveness of human and murine cells to ΔS Luc-GFP replication. The indicated human and murine cells were infected with ΔS-VRP(G), and at 18 hpi, luciferase activity and GFP expression were measured. (A and B) Luciferase expression in human and murine cell lines. (C and D) GFP expression in human and murine cell lines. (E and F) Primary BMDM and BMDC were infected with ΔS-VRP(G), and at 18 hpi, expression levels of viral N mRNA and host antiviral genes were measured by qRT-PCR. (E) Viral N mRNA expression; (F) host antiviral gene expression.

FIG 4

The ΔS-VRP(G) reporter system is suitable for antiviral drug screening. Huh7.5 or A549 cells were infected with ΔS-VRP(G) virus, and at 2 hpi, infected cells were treated with the indicated concentrations of remdesivir or GC376 dissolved in DMSO. At 18 hpi, GFP expression and luciferase activity were measured. (A and B) Assessment of effects of remdesivir treatment on ΔS Luc-GFP replication in Huh7.5 and A549 cells. (C and D) Assessment of effects of GC376 treatment on ΔS Luc-GFP replication in Huh7.5 and A549 cells. Luciferase values are normalized to DMSO control and shown as a percentage of DMSO control.