Remdesivir Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC₅₀ = 0.01 μM). Weaker activity is observed in Vero E6 cells (EC₅₀ = 1.65 μM) because of their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase of SARS-CoV-2. In mice infected with the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with vehicle-treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.

Keywords: COVID-19; GS-441524; RNA-dependent RNA polymerase; RdRp; SARS-CoV-2; antiviral; coronavirus; mouse; remdesivir; therapeutic.

Graphical abstract

Figure 1

Modeling of RDV onto the SARS-CoV-2 RdRp Structure (A) Model of the SARS-CoV-2 polymerization complex in its elongating state. The model was based on the cryo-EM apo structures of SARS-CoV (http://www.rcsb.org/structure/6NUR) and SARS-CoV-2 (http://www.rcsb.org/structure/6M71). The active site is boxed in red. (B) Enlarged view of the active site, depicting RDV pre-incorporation. The 1′CN substituent sits in a shallow pocket formed by residues T687 and A688. Bound to the two catalytic Mg²⁺ ions (pink), the TP is coordinated by two basic residues (R553 and R555). See also Figures S1–S3.

Figure 2

The Prodrug Remdesivir (RDV) and Parent Nucleoside GS-441524 (’524) Potently Inhibit SARS-CoV-2 Replication (A) Vero E6, Vero CCL-81 (Vero), Huh7, and Calu3 2B4 cells were infected with an MOI of 0.01 and/or 0.1 PFU/cell SARS-CoV-2 (2019-nCoV/USA-WA1/2020), and infectious viral titers were determined by plaque assay 0.5, 24, 48, and 72 h post-infection (hpi). Viral yields were calculated by subtracting the average 0.5 h (post-adsorption, pre-incubation) titer from each subsequent time point. Data represent the average of three replicates from one experiment. Error bars indicate SD. B.D., below detection. Calu3 2B4 cells were infected with 0.1 PFU/cell SARS-CoV-2, and Vero cells were infected with 0.01 PFU/cell SARS-CoV-2 and treated with RDV, GS-441524 (’524), or DMSO only (control) in cell culture medium. Supernatants were collected 48 hpi (Vero E6 cells) or 72 hpi (Calu3 2B4 cells). (B and C) Reduction of SARS-CoV-2 replication by RDV in Calu3 2B4 cells, as determined by infectious viral titer (B) and qRT-PCR (C). (D) Percent inhibition of SARS-CoV-2 replication by RDV and GS-441524 in Calu3 2B4 cells, as determined by infectious viral titer (RDV: EC₅₀ = 0.28 μM, EC₉₀ = 2.48 μM; GS-441524: EC₅₀ = 0.62 μM, EC₉₀ = 1.34 μM). No significant cytotoxicity of either compound was detected in Calu3 cells. (E) Percent inhibition of SARS-CoV-2 replication by RDV and GS-441524 in Calu3 2B4 cells, as determined by qRT-qPCR (RDV: EC₅₀ = 0.60 μM, EC₉₀ = 1.28 μM; GS-441524: EC₅₀ = 1.09 μM, EC₉₀ = 1.37 μM). (F and G) Reduction of SARS-CoV-2 replication by RDV in Vero E6 cells, as determined by infectious viral titer (F) and qRT-PCR (G). (H) Percent inhibition of SARS-CoV-2 replication by RDV and GS-441524 in Vero E6 cells, as determined by infectious viral titer (RDV: EC₅₀ = 1.65 μM, EC₉₀ = 2.40 μM; GS-441524: EC₅₀ = 0.47 μM, EC₉₀ = 0.71 μM). No significant cytotoxicity of either compound was detected in Vero E6 cells. (I) Percent inhibition of SARS-CoV-2 replication by RDV and GS-441524 in Vero E6 cells, as determined by qRT-PCR (RDV: EC₅₀ = 1.49 μM, EC₉₀ = 3.03 μM; GS-441524: EC₅₀ = 0.47 μM, EC₉₀ = 0.80 μM). Data represent means of 2–4 independent experiments with 2–3 replicates each. Error bars represent SEM. See also Figure S5.

Figure 3

RDV Is Potently Antiviral against SARS-CoV-2 in Primary Human Airway Epithelial (HAE) Cultures HAE cultures were infected with a SARS-CoV-2 clinical isolate (2019-nCoV/USA-WA1/2020) at an MOI of 0.5 PFU/cell for 2 h, and then the virus was removed and cultures were washed 3 times, followed by incubation at 37°C for 48 h. (A) SARS-CoV-2 infectious virus production in two independent studies. The virus was titered via plaque assay in apical washes 48 hpi. Each symbol represents the titer from a single culture, and a line is drawn at the mean. (B) Percent inhibition generated from the titer data in (A). See also Figure S4.

Figure 4

RDV-TP Levels in Vero E6, Calu3, and HAE Cultures Vero E6 cells, Calu3 2B4 cells, and HAE cultures were incubated with RDV or GS-441524. At 8, 24, and 48 h of treatment, whole-cell extracts were prepared, and RDV-TP levels were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described in STAR Methods. RDV-TP levels in Vero E6 and Calu3 2B4 cells represent mean ± SD from n = 2 or 4 independent experiments, respectively, each performed with duplicate samples. RDV-TP levels in HAEs represent the mean ± SD of four replicates for each individual donor (D1 and D2). See also Tables S1 and S2 and Figure S5.

Figure 5

RDV Is Active against the SARS-CoV-2 RdRp In Vivo The activity of RDV against the SARS-CoV-2 RdRp was evaluated using a chimeric SARS-CoV encoding the SARS-CoV-2 RdRp (SARS/SARS2-RdRp). (A) Schematic of the recombinant SARS-CoV mouse-adapted MA15 strain chimeric virus genomes generated for these studies. SARS/SARS2-RdRp and SARS/SARS2-RdRp-nanoluciferase (nLUC) were constructed by exchanging the SARS-CoV MA15 RdRp with the SARS-CoV-2 RdRp. ORF7 is replaced by nLUC in SARS2-RdRp-nLUC. (B) The presence of the SARS-CoV-2 RdRp was confirmed by Sanger sequencing in stocks of both recombinant chimeric viruses. Alignment of a stretch of nucleotides from the SARS-CoV and SARS-CoV-2 RdRp highlighting nucleotides that differ between the two strains is shown in boldface. These SARS-CoV-2 RdRp-specific nucleotides are present in both chimeric viruses used in this study, as determined by Sanger sequencing and shown in the histogram. (C) SARS/SARS2-RdRp-nLUC replication in Huh7 cells in the presence of RDV (left) and associated percent inhibition (right). (D) SARS-CoV replication in Huh7 cells in the presence of RDV (left) and associated percent inhibition (right). (E) Percent starting weight of 17-week-old female Ces1c^−/− mice infected intranasally with 1 × 10³ PFU of SARS/SARS2-RdRp and treated subcutaneously with 25 mg/kg RDV or vehicle 1 day post-infection (dpi) and twice daily thereafter. (F) Lung hemorrhage at 5 dpi. (G) Pulmonary function by WBP. The PenH metric shown is a surrogate marker of pulmonary obstruction. p < 0.0001 as determined by two-way ANOVA with Sidak’s multiple comparisons test. (H) Lung titer at 5 dpi as measured by plaque assay. p = 0.0012 by Mann-Whitney test. In (E) and (G), boxes encompass the 25^th–75^th percentile, a line is drawn at the median, and whiskers represent the range.