A Novel Viral Assembly Inhibitor Blocks SARS-CoV-2 Replication in Airway Epithelial Cells

Abstract

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for novel therapies with high genetic barriers to resistance. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Our data demonstrate that PAV-104 inhibited > 99% of infection with diverse SARS-CoV-2 variants in primary and immortalized human AECs. PAV-104 suppressed SARS-CoV-2 production without affecting viral entry or protein synthesis. PAV-104 interacted with SARS-CoV-2 nucleocapsid (N) and interfered with its oligomerization, blocking particle assembly. Transcriptomic analysis revealed that PAV-104 reversed SARS-CoV-2 induction of the Type-I interferon response and the 'maturation of nucleoprotein' signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19.

Figure 1. Synthesis and molecular structure of PAV-104.

A detailed description of PAV-104 synthesis and preparation is provided in the Methods section.

Figure 2. PAV-104 decreases virus production in SARS-CoV-2-infected Calu-3 cells.

(A) MTT assay was performed on Calu-3 cells to examine the cellular toxicity of PAV-104. Relative cell viability was displayed based on the PAV-104-untreated control (set at 100%). The red arrow represents the CC₅₀ value (3732 nM) of PAV-104. (B) Anti-SARS-CoV-2 activity of PAV-104 in Calu-3 cells was measured by RT-qPCR targeting the N genes. Cells were pretreated with PAV-104 at the indicated concentrations for 1 hour, followed by infection with SARS-CoV-2 (MOI=0.01) for 24 h in the presence of PAV-104. RNA isolation and RT-qPCR assay was performed 24 h post-infection. (C) The SARS-CoV-2 titer (TCID₅₀) was measured after treatment with varying doses of PAV-104 as described in (B). (D) Immunofluorescence staining of Calu-3 cells with DAPI (blue) was performed at 72 h postinfection. Cells were pretreated with PAV-104 at the indicated concentrations, followed by infection with SARS-CoV-2-GFP virus. Scale bar, 500 μm. (E) Quantification of SARS-CoV-2 infected cells (GFP positive cells) in Calu-3 cells (shown in panel D). Data are representative of the results of three independent experiments (mean ± SEM). Statistical significance was analyzed by t test. p≤0.05 [*], p ≤ 0.01 [**], p≤0.001 [***], p≤0.0001 [****].

Figure 3. PAV-104 inhibits SARS-CoV-2 replication in Calu-3 cells more potently than remdesivir.

(A) Reduction of SARS-CoV-2 replication by PAV-104 and remdesivir in Calu-3 cells, as determined by RT-qPCR targeting the N gene. Calu-3 cells were pretreated with DMSO, PAV-104, or remdesivir for one hour, then infected with SARS-CoV-2 at an MOI of 0.001. Supernatants and cells were collected at 48 hpi. (B) Percent inhibition of SARS-Cov-2 replication by PAV-104 and remdesivir in Calu-3 cells, as determined by RT-qPCR (PAV-104: EC₅₀ = 1.7 nM, EC₉₀ = 23.5 nM; remdesivir: EC₅₀ = 7.9 nM, EC₉₀ = 218.1 nM). (C) Reduction of SARS-CoV-2 replication by PAV-104 and remdesivir in Calu-3 cells, as determined by infectious viral titer. (D) Percent inhibition of SARS-CoV-2 replication by PAV-104 and remdesivir in Calu-3 cells, as determined by infectious viral titer (PAV-104: EC₅₀ = 0.5 nM, EC₉₀ = 10.3 nM; remdesivir: EC₅₀ = 0.65 nM, EC₉₀ = 19.2 nM). Data are representative of the results of three independent experiments (mean ± SEM).

Figure 4. PAV-104 inhibits the replication of SARS-CoV-2 variants in human primary airway epithelial cells.

(A) Antiviral activity of PAV-104 against SARS-CoV-2 in primary AECs, as determined by RT-qPCR. ALI-cultured primary AECs were pre-incubated with DMSO or PAV-104 at indicated concentrations for one hour and were then infected with heat-inactivated virus and SARS-CoV-2 (lineage P.1, MOI=0.1) at the apical and basal compartment for two hours. Cells were then washed and supplemented with fresh media containing DMSO or PAV-104. Cells were collected for RNA isolation and RT-qPCR at 36 hpi. Each color represents data from one donor. (B) Antiviral activity of PAV-104 against SARS-CoV-2 variants (Delta and Omicron) in primary AECs, as determined by RT-qPCR. Each color represents data from one donor. Data are representative of the results of three independent experiments (mean ± SEM). Statistical significance was analyzed by paired t tests. p≤0.05 [*], p ≤ 0.01 [**], p≤0.001[***], p≤0.0001 [****].

Figure 5. PAV-104 inhibits SARS-CoV-2 replication at a post-entry step of the viral life cycle.

(A) Schematic timeline of PAV-104 treatment in Calu-3 cells. Calu-3 cells were incubated with PAV-104 or infected with SARS-CoV-2 at indicated time points as the diagram shows. (B) Virus production (measured as viral titer) in Calu-3 cells treated with PAV-104 at indicated doses and time points. (C) Schematic timeline of PAV-104 treatment in ALI-cultured primary AECs. Primary AECs were treated with PAV-104 or infected with SARS-CoV-2 at indicated time points. (D) Virus production (measured as viral N gene expression by RT-qPCR) in primary AECs treated with PAV-104 at indicated doses and time points. Heat-inactivated SARS-Cov-2 treatment was used for normalization. Data are representative of the results of three independent experiments (mean ± SEM). Statistical significance was analyzed by t test or paired t test. p≤0.05 [*], p≤0.01 [**], p≤0.001[***], p≤0.0001 [****].

Figure 6. PAV-104 blocks SARS-CoV-2 viral assembly/budding.

(A) Western blot analysis of structural protein expression in cell lysates and ultracentrifuged pellets. HEK293T cells were transfected with plasmids encoding the proteins indicated at the top. Western blots were performed with the primary antibodies indicated on the left of the blots. Anti-β-actin antibody was used as a loading control. (B and C) Relative quantification of the indicated protein from western blot (A). β-actin was used as a loading control for cell lysates and pellets. (D) Quantification of SARS-CoV-2 VLPs by nanoparticle tracking analysis. HEK293T cells were transfected with plasmids encoding the proteins indicated at the top. VLPs containing nanoparticles in the ultracentrifuged pellets from cell culture supernatants were diluted to a concentration in the range of 10⁷–10⁹/ml and examined using a NanoSight NS300 (NanoSight, Ltd) equipped with a 405 nm laser. Data are representative of the results of three independent experiments (mean ± SEM). Statistical significance was analyzed by t test or paired t test. p≤0.05 [*], p≤ 0.01 [**], p≤0.001 [***], p≤0.0001 [****]

Figure 7. PAV-104 interacts with N and interferes with N oligomerization.

(A) Quantitation of resin-bound N band density detected by western blot. DRAC experiments were performed on the PAV-104 resin column in triplicate and control resin column in singlicate from cell extracts prepared from Calu-3 cells that were uninfected (Un-Inf) or infected with SARS-CoV-2 Delta variant (Delta) or SARS-CoV-2 Omicron variant (Omicron). Material bound to the PAV-104 resin was run on gels and western blot for SARS-CoV-2 N. (B) Concentration of SARS-CoV-2 N in each fraction. Cell extracts from N-transfected cells in the presence or absence of PAV-104 were sedimented in a 10–40% glycerol gradient at 135000 g for 20 hours. Twenty-two fractions were collected and protein content analyzed using a commercial SARS-CoV-2 N protein sandwich ELISA kit (duplicate). LD=cell extracts without sedimentation. Data are representative of the results as mean ± SEM. Statistical significance was analyzed by t test. p≤0.05 [*], p≤ 0.01 [**], p≤0.001 [***], p≤0.0001[****].

Figure 8. Impact of SARS-CoV-2 infection and PAV-104 treatment on the transcriptome of primary AECs.

(A-C) Volcano plots showing the proportion of differentially-expressed genes (DEGs) in the setting of SARS-CoV-2 infection (SARS-CoV-2 infection vs Control (SC)) (A), SARS-CoV-2 infection in the presence of PAV-104 (SARS-CoV-2 infection+PAV104 vs Control (PC)), and SARS-CoV-2 infection in the presence of PAV-104 vs SARS-CoV-2 infection (PS). DEGs (FDR<0.05) with log2(fold change) > 0.5 are indicated in red. DEGs (FDR<0.05) with log2(fold change) < −0.5 are indicated in blue. The absolute value of Log2(fold change) < 0.5 and non-significant DEGs are indicated in gray. (D) Top enriched REACTOME pathways in response to SARS-CoV-2 infection or PAV-104 treatment identified using gene set enrichment analysis (GSEA). The orange and blue-colored bars in the bar chart indicate predicted pathway activation or predicted inhibition, respectively, based on enrichment-score. Y represents FDR < 0.25. (E) Sample coverage tracks from the QIAGEN genome browser depicting SARS-CoV-2 assembly. Mapped read counts of Control, SARS-CoV-2 infection, and SARS-CoV-2 infection in the presence of PAV-104 (SARS-CoV-2 infection+PAV-104) are 0 to 3, 0 to 392760, and 0 to 1790, respectively.