Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CLpro Reporter Assay

Abstract

In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CL^pro 3CL^pro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CL^pro Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CL^pro This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread.IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CL^pro This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.

Keywords: FlipGFP; antivirals; coronavirus; protease; screening.

FIG 1

A FlipGFP protease reporter with coronavirus cleavage sites fluoresces after SARS-CoV-2 3CL^pro expression. (A) Diagram of the FlipGFP protease reporter (16) with coronavirus cleavage sequences. FlipGFP splits GFP into β1-9 and β10-11, with β11 held in parallel to β10 by heterodimerized coiled coils E5/K5 and a linker sequence containing a coronavirus cleavage site. The CoV main protease, 3CL^pro, cuts at the cleavage site, allowing β11 to “flip” antiparallel to β10, enabling self-assembly of the complete GFP beta-barrel and resulting in detectable fluorescence. The pan-coronavirus 3CL^pro consensus sequence, LQ, is in bold. (B) Quantification of fluorescence from 293T cells 48 h after transfection with each FlipGFP reporter or superfolder GFP (sfGFP) individually and without a protease. Statistical analysis is relative to sfGFP. (C) Quantification of fluorescence from 293T cells 48 h after transfection with each FlipGFP reporter and either the SARS-CoV-2 3CL^pro or an influenza virus protein (A/PR8/1834 NP). Statistical analysis is relative to NP control. (D) Images corresponding to panel C. Green, cleaved FlipGFP; blue, nuclei. Data are shown as means ± SDs (n = 3). P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001; ns, not significant). Experiments were performed twice.

FIG 2

Conservation of coronavirus 3CL^pro activity enables CoV protease reporter compatibility with many coronaviruses. (A) Phylogenetic tree of five coronaviruses, SARS-CoV-2, SARS-CoV, murine hepatitis virus (MHV), avian infectious bronchitis virus (IBV), and HCoV-229E, generated based on the polyprotein ORF1ab using NCBI Virus (34). These viruses span three coronavirus groups: Alphacoronavirus, Betacoronavirus, and Gammacoronavirus. 3CL^pro protein sequence identities are compared to the SARS-CoV-2 3CL^pro. (B) Microscopy of 293T cells 48 h after transfection with CoV reporter 3 and coronavirus 3CL^pro proteins or an influenza virus protein (A/PR8/1834 NP). Green, cleaved FlipGFP; blue, nuclei. (C) In black is quantification of the data in panel B. In blue are results of qPCR of CoV 3CL^pro or PR8 NP RNA levels relative to untransfected cells. Data are shown as means ± SDs (n = 3); statistical analysis is relative to the NP control. P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001). Experiments were performed twice.

FIG 3

Inhibition of the SARS-CoV-2 3CL^pro by the protease inhibitor GC376 is measurable with the fluorescent CoV protease reporter. (A) Microscopy of 293T cells before or 12, 24, and 48 h after transfection with CoV reporter 3 and SARS-CoV-2 3CL^pro. Green, cleaved FlipGFP; blue, nuclei. (B) Quantification of data in panel A. Data are shown as means ± SDs (n = 3); statistical analysis is relative to the NP control. P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001). (C) Quantification of 293T cells 24 h after transfection with CoV reporter 3 and SARS-CoV-2 3CL^pro, with decreasing levels of 3CL^pro. Data are shown as means ± SDs (n = 3); statistical analysis is relative to a 1:1 ratio of reporter to protease. P values were calculated using unpaired, two-tailed Student’s t tests (*, P < 0.05; **, P < 0.001). (D) In black is quantification of 293T cells 24 h after transfection with CoV reporter 3 and SARS-CoV-2 3CL^pro and treatment with the pan-coronavirus protease inhibitor GC376. Data are shown as means ± SDs with nonlinear fit curve (n = 3). In blue is calculation of cell viability relative to vehicle-only (DMSO) samples. (E) In black are results of RT-qPCR of VeroE6 cells 24 h after infection with SARS-CoV-2 at an MOI of 0.01 and treatment with the pan-coronavirus protease inhibitor GC376. Data are shown as means ± SDs with nonlinear fit curve (n = 4). In blue is calculation of cell viability relative to vehicle-only (DMSO) samples. Data are shown as means ± SDs (n = 3). Experiments were performed twice.