A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC ₅₀ values were much higher than that required for HIV-1. Taken together, this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad number of RNA and DNA viruses.

Fig 1.. Development of a facile Q-RT-PCR assay for SARS-CoV-2 viral RNA detection in cell culture supernatants.

(A) Serially diluted RNA standards were either directly subjected to Q-RT-PCR or processed as in the modified protocol detailed in the text prior to Q-RT-PCR. Log₂ (copies) are plotted against the cycle threshold (Ct) values. Linear regression analysis was done to obtain the equations. Data show the average of three independent biological replicates. Error bars show the SEM. (B) Comparison of the efficiency and detection ranges for quantifying SARS-CoV-2 RNA using purified RNA or lysed supernatants from virus stocks. Data are derived from three independent replicates. Error bars show the SEM. (C) Vero E6 cells were infected at an MOI of 0.01 and cell culture supernatants were analyzed for SARS-CoV-2 RNA following the conventional RNA extraction protocol vs. the modified protocol developed herein at various times post infection. Cell-associated viral RNA was analyzed in parallel following RNA extraction for reference. Data are from three independent biological replicates. Error bars show the SEM. (D) Illustration of the efficiency and detection ranges of Taqman-based and SYBR-Green-based Q-RT-PCR quantifying known amounts of SARS-CoV-2 RNA. Data is from 2–3 independent replicates. Error bars show the SEM.

Fig 2.. A compound screen to validate SARS-CoV-2-specific inhibitors and entry pathways.

Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.01 and inhibitors were added concomitantly at concentrations shown in the figures following virus adsorption. Supernatants from infected cells were lysed and used in a SYBR-Green based Q-RT PCR to quantify the viral RNA in cell culture supernatants. Compound cytotoxicity was monitored by RealTime-Glo™ MT Cell Viability Assay Kit (Promega) in parallel plates. Data show the cumulative data from 2–5 independent biological replicates. Error bars show the SEM.