key: cord-1041697-n1h4zzs7
authors: Uppal, Timsy; Tuffo, Kai; Khaiboullina, Svetlana; Reganti, Sivani; Pandori, Mark; Verma, Subhash C.
title: Screening of SARS-CoV-2 Antivirals Through a Cell-Based RNA-Dependent RNA Polymerase (RdRp) Reporter Assay
date: 2022-04-05
journal: bioRxiv
DOI: 10.1101/2022.04.04.486994
sha: 4c2b12dc876067b5de0fb3fefd6d60b2a9e064d7
doc_id: 1041697
cord_uid: n1h4zzs7

COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose international public health threat and thus far, has resulted in greater than 5.6 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast spreading COVID-19 variants that may elude vaccines efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using a known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5’CT, and dasabuvir. Among these inhibitors, dasabuvir (FDA-approved drug) exhibited promising RdRp inhibitory activity. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with IC50 values 9.47 μM and 10.48 μM, for USA-WA1/2020 and B.1.617.2 variants, respectively). Our results suggests that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. In addition, our assays provide robust, target-specific, and high-throughput screening compatible (z- and z’-factors of > 0.5) platforms that will be a valuable tool for the screening SARS-CoV-2 RdRp inhibitors. Significance SARS-CoV-2 has caused a major public crisis world has seen in recent history. Development of vaccines and emergency use authorization of anti-virals are helping in reducing the burden of SARS-CoV-2 caused hospitalization and deaths. However, there is still need for optimal anti-viral(s) that can efficiently block viral propagation, and targeting viral polymerase (RdRp) is an among the most suitable targets for clamping viral replication. In this study, we developed a cell-based assay to screen potential compounds capable of blocking RdRp activity. The efficacy of our assay was validated by using already approved anti-virals, which reduced RdRp activity and slowed the replication of two SARS-CoV-2 variants (WA1 USA-WA1/2020 and B.1.617.2) in a cell culture model. This confirmed that our system can be used for identifying potential anti-SARS-CoV-2 anti-virals.

plate. For compound treatment, cells were treated with selective inhibitors at a final conc. of 10 µM or 123 DMSO alone (control) for 48h. Cells were then lysed in 100uL of 1X passive lysis buffer and incubated at 124 RT for 15 min to ensure complete lysis. The lysate was transferred to a 96-well microplate, followed by 

For relative viral genome quantification post infection and compound treatment, control or USA-WA1/2020 164 virus was added onto the Vero E6 cells plated in a 24-well plate (100,000 cells per well) for 2h (37 °C and 165 5% CO2). Post infection, the virus containing medium was replaced with 1 mL fresh medium containing 166 either DMSO (control) or compounds (10µM). The cells were treated with compounds for 72h at 37 °C and 167 5% CO2. For the detection of viral genomic RNA through qRT-PCR, supernatant from control or compound- Cell toxicity due to the compound treatment was determined using the standard colorimetric MTT [3-(4,5- 176 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Thermo Fisher Scientific) according to the 177 manufacturer's instructions. Briefly, 5,000 cells resuspended in the complete DMEM medium were seeded 178 in a costar 96-well microplate and grown overnight. Next day, cells were washed with 1X PBS and treated culture medium (100µL) was used as a negative control (blank). After 4h, when intracellular purple . FLuc gene is flanked by 5'-UTR and 3′-UTR of SARS-CoV-2 and the hepatitis delta virus (HDV) ribozyme self-cleavage sequence. RLuc serves as an internal control to normalize FLuc activity. Expression of RdRp in trans will control FLuc levels and compounds/mutations altering RdRp activity will be reflected by reduction in FLuc levels. (B) Schematic representation of the plasmid construct expressing SARS-CoV-2 RdRp (nsp12), accessory proteins (nsp7, and nsp8) and viral 3CL pro protease (nsp5), required for proper cleavage of the proteins. Cleavage of the proteins is confirmed by the expression of flag-tagged nsp12 (RdRp) in an immunoblotting assay.

(RdRp) helped in determining the expression and cleavage from the polypeptide.

To test our SARS-CoV-2 RdRp reporter system and the expression and the activity intracellular transfection, we tested the expression of flag-tagged SARS-CoV-2 RdRp in the cells by western blotting Cells stably expressing the SARS-CoV-2 RdRp reporter and pcDNA3.1 or flag-tagged nsp5,7,8, and 12 constuct were mock (DMSO) treated or treated with remdesivir (a potent anti-RdRp inhibitor), followed by luminescence detection with the dual luciferase reporter assay, and the relative FLuc/RLuc ratio (Index) was detected after 48h. (B) Cells were also subjected to immunoblotting with anti-flag antibody 48h post-transfection, and C) Total RNA was extracted and relative FLuc/RLuc RNA expression levels were quantified by RT-qPCR, using luciferase gene specific primers and beta-actin, as an internal control. luciferase reporter assay. In addition, we treated equal number of 293-RdRp-nsp5, 7, 8,12-flag cells with 287 remdesivir, a known SARS-CoV-2 RdRp inhibitor, to further validate our SARS-CoV-2 RdRp reporter 288 system. As seen in Figure 3A , the calculated FLuc/RLuc ratio (Index value) in the cells constitutively 1 and 2). The levels of (+) FLuc RNAs were also quantified after treatment with remdesivir. As seen 302 in Figure 3C , treatment with RdRp inhibitor significantly reduced the mRNA levels of (+) FLuc. This 303 confirmed that we have developed a cell-based reporter system to quantitatively assess the intracellular 304 SARS-CoV-2 RdRp activity.

To determine if the cell-based reporter assay is suitable for HTS applications, we calculated 309 screening window coefficients, Z-, and Z'-factors using Zhang's formulae (19). The Z-, and Z'-factors are Figure 4 . Evaluation of HEK293 cell-based SARS-CoV-2 RdRp Reporter System for HTS assays. The experimental groups were characterized as: control group (n=24)-cells expressing SARS-CoV-2 RdRp reporter and pcDNA3.1 plasmids treated with DMSO (0.025%); positive group (n=24)-cells expressing SARS-CoV-2 RdRp reporter and flag-tagged nsp5, 7, 8, and 12 plasmids treated with DMSO (0.025%); and inhibitor group (n=24)-cells expressing SARS-CoV-2 RdRp reporter and flag-tagged nsp5, 7, 8, and 12 plasmids treated with remdesivir (10µM). The Z-and Z'-factors were calculated for HEK293 cell-based SARS-CoV-2 RdRp reporter systems, using control and positive, and positive and inhibitor groups in each 24-well plate. According to Zhang's formula, the Z-and Z'factors for HEK293 cell based reporter assay were calculated as 0.732, and 0.789, respectively, indicating assay system has the required robustness and reproducibility for HTS extracted and FLuc mRNA and RLuc mRNA expression levels were assayed by qRT-PCR. without and with remdesivir (10µM, inhibitor group) treatments for 24h thrugh dual luciferase reporter 316 assay. The Z-factor (specificity of the assay for SARS-CoV-2 RdRp activity) and Z′-factor (applicability of 317 remdesivir as a positive control), were calculated using the relative FLuc activity obtained from control and 318 positive, and positive and inhibitor groups, respectively. We obtained a Z-factor and Z′-factor values of 319 0.732 and 0.789 respectively, indicating the HEK293 cell-based reporter assay is excellent for assaying RdRp 320 activity of SARS-CoV-2, and confirming the use of remdesivir as a positive control for SARS-CoV-2 RdRp 321 specific inhibitors in HTS assays ( Figure 4) . Thus, the target-specific HEK293 cell-based assay system 322 targeting the RdRp of SARS-CoV-2 shall provide excellent platform to facilitate identification of efficacious 323 SARS-CoV-2 RdRp antivirals.

We further evaluated if the optimized SARS-CoV-2 RdRp reporter system allows for the screening of whether these antivirals have cytotoxic effects on those treated cells, which may also contribute to alter 342 levels of FLuc/RLuc, we performed cell viability assay using MTT analysis. This colorimetric assay provides 343 a sensitive and accurate method for the determination of cell viability based on the generation of an 344 insoluble formazan (purple color) from water-soluble tetrazolium MTT dye. As shown in Figure 5C , no 345 detectable cytotoxic effects were observed in the MTT analysis, when the cells were treated with 10µM 346 conc. of these selected anti-virals for 48h. Hence, this indicated that our HEK293 cell-based SARS-CoV-2 347

RdRp reporter system will be instrumental in screening additional novel inhibitors of SARS-CoV-2 RdRp 348 activity.

We further investigated the antiviral activity of chosen compounds against SARS-CoV-2 replication in vitro.

To this end, USA-WA1/2020 (1.6 X 10 6 TCID50/mL; Lot # 70036318) and B.1.617.2 (6.5 X 10 5 TCID50/mL 2 ;

Lot # 70045238) variants were obtained from BEI Resources and propagated in Vero E6 cells. Viral titers 

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