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

doi:10.21203/rs.3.rs-2887435/v1

Download PDF

Article

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

https://doi.org/10.21203/rs.3.rs-2887435/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 22 Apr, 2024

Read the published version in Communications Biology →

Version 1

posted

You are reading this latest preprint version

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.

Biological sciences/Microbiology/Antimicrobials/Antiviral agents

Biological sciences/Microbiology/Virology/SARS-CoV-2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, belongs to a highly contagious betacoronavirus¹. The high transmission and variation of SARS-CoV-2 poses an ongoing threat to global public health. Despite multiple vaccine options, only 65.4% of people are currently fully vaccinated worldwide, due in part to lack of vaccine access as well as behavioral resistance to vaccination. Moreover, many people remain at high risk for severe COVID-19 due to decreased vaccine efficacy and increased risk of respiratory failure associated with immune compromise.

For the treatment of SARS-CoV-2, anti-SARS-CoV-2 monoclonal antibodies (mAbs) that target the spike protein represent one class of therapeutic candidates approved by the FDA for COVID-19 patients^2,3. However, the efficacy of anti-SARS-CoV-2 mAbs is negligible in the face of currently circulating viral variants⁴. Beyond mAbs, antiviral small-molecule drugs have been developed that target specific parts of the viral life cycle to prevent infectivity, severe illness and death attributed to COVID-19. Three antiviral agents are currently authorized by the FDA for the treatment of COVID-19: viral RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir and molnupiravir^4–6 and a viral 3C-like protease inhibitor, paxlovid, which consists of nirmatrelvir and ritonavir⁷. Clinical studies have shown that remdesivir is not associated with statistically significant clinical benefits⁸. In vitro studies have shown that molnupiravir is used as a substrate by host RNA polymerases including the mitochondrial DNA-dependent RNA polymerase⁹. Paxlovid treatment is often associated with COVID-19 rebound following the treatment cycle¹⁰. Taken together, these realities illustrate the need for effective and broad-spectrum antiviral drugs for COVID-19 with minimal off-target effects.

Recent studies have focused on the SARS-CoV-2 viral life cycle to find additional targets for drug therapy, providing candidate SARS-CoV-2 entry and attachment inhibitors^11,12, novel viral protease inhibitors^13,14, and N assembly inhibitors that reduce viral nucleocapsid (N)-genome RNA interaction^15,16. However, data are limited describing agents that selectively inhibit the late stage of the SARS-CoV-2 viral life cycle of particle formation/budding¹⁷. The viral assembly/budding process is a dynamic program dependent on transient multi-protein assembly complexes¹⁸. One approach to identifying these host-viral drug targets is by interrogation of the pathway of viral assembly/budding in cell-free protein synthesis and assembly systems^19–21. Small molecules have been identified in this manner that are active against various viral families including those of rabies virus, HIV-1, and influenza virus^22–24.

The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis and assembly (CFPSA), was recently shown to target host protein assembly machinery in a manner specific to viral assembly²³. This compound has minimal host toxicity, and once-daily oral dosing in rats that achieves > 200-fold of the 90% effective concentration (EC₉₀) in blood. Pharmacokinetic studies of PAV-104 in rats showed a lung to plasma ratio of 0.3 after oral administration²³. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape.

Here, we evaluated the antiviral effect of PAV-104 against SARS-CoV-2 infection in immortalized and primary human airway epithelial cells (AECs), and elucidated the mechanism underlying this antiviral activity. Our results show that PAV-104 treatment potently inhibits the replication of diverse SARS-CoV-2 variants. We further demonstrate that PAV-104 specifically inhibits the late stages of the SARS-CoV-2 replication cycle, perturbing the oligomerization of viral N, thereby blocking viral capsid assembly and budding. PAV-104 treatment also reverses SARS-CoV-2 induction of the Type-I interferon response and the ‘maturation of nucleoprotein’ signaling pathway which supports coronavirus replication within the target cell. Together, our data suggest that PAV-104 is a promising therapeutic candidate for COVID-19, and its mechanism of action is distinct from existing clinical strategies.

PAV-104 suppresses SARS-CoV-2 infection in Calu-3 cells

The synthesis and molecular structure of PAV-104 (Fig. 1), a small molecule drug recently associated with pan-respiratory virus antiviral activity²³, is described in detail in the Methods section. To investigate the impact of PAV-104 on SARS-CoV-2 infection, we first determined the cytotoxicity of PAV-104, to optimize dosing in Calu-3 cells. The 50% cytotoxic concentration (CC₅₀) value detected by MTT assay was 3732 nM for PAV-104 in Calu-3 cells (Fig. 2A). Next, we evaluated the effects of PAV-104 on SARS-CoV-2 replication at 6.25 nM, 25 nM, 50 nM, and 100 nM concentrations. Calu-3 cells were pretreated for one hour with PAV-104 followed by infection with SARS-CoV-2 at an MOI of 0.01, and PAV-104 was maintained in the media until 24 hours following viral infection. SARS-CoV-2 replication, as measured by quantitation of viral nucleocapsid (N) gene expression, was decreased significantly by treatment with PAV-104 in a dose dependent manner (p < 0.01) (Fig. 2B). Similarly, release of infectious virus in the supernatant was suppressed significantly by PAV-104 in a dose dependent manner, as measured by median tissue culture infectious dose (TCID₅₀) (p < 0.01) (Fig. 2C), with up to 75-fold reduction at the highest concentration of PAV-104. The inhibition of virus production by PAV-104 was confirmed by specific staining of viral N using an immunofluorescence assay (IFA) (Fig. 2D, E). Taken together, these data demonstrate that PAV-104 decreases SARS-CoV-2 viral production in susceptible Calu-3 cells.

PAV-104 and remdesivir potently inhibit SARS-CoV-2 replication

Remdesivir is the first approved small-molecule anti-SARS-CoV-2 drug for COVID-19 treatment and shows potent anti-SARS-CoV-2 activity in Calu-3 cells^25,26. To compare the anti-SARS-CoV-2 efficacies of PAV-104 with remdesivir in Calu-3 cells, Calu-3 cells were infected with SARS-CoV-2 for 48 hours at the MOI of 0.001 and treated with varying doses of PAV-104 or remdesivir. Cells and supernatants were harvested for quantification of N expression by RT-qPCR and quantification of infectious viral titers by TCID₅₀. Both compounds displayed dose-dependent inhibition of viral replication (Fig. 3A, B, C, D). Remdesivir inhibited SARS-CoV-2 with an EC₅₀ value of 7.9 nM and a 90% maximal effective concentration (EC₉₀) value of 218.1 nM (Fig. 3B). PAV-104 was more potent (EC₅₀ = 1.7 nM and EC₉₀ = 23.5 nM) than remdesivir, as determined by RT-qPCR (p < 0.05) (Fig. 3B). EC₅₀ value determined by quantification of infectious virus in the supernatants showed a similar trend (Fig. 3D). Thus, PAV-104 inhibits SARS-CoV-2 more potently than remdesivir in Calu-3 cells.

PAV-104 is a highly potent antiviral inhibitor of SARS-CoV-2 in primary airway epithelial cells

Upper and lower airways in humans are known to be the first gateway for SARS-CoV-2 infection²⁷. To investigate the antiviral activity of PAV-104 against SARS-CoV-2 in human primary airway epithelial cells (AECs), we performed antiviral assays in air/liquid interface (ALI)-cultured AECs, which is useful in modeling the in vivo effects of PAV-104 on SARS-CoV-2 infection ex vivo²⁸. We pretreated primary AECs from three healthy donors with PAV-104 and then infected them with the SARS-CoV-2 Gamma variant (Pango lineage designation P.1) for 36 hours. In PAV-104-treated, SARS-CoV-2-infected AECs cultures, there was > 99% inhibition of infection with PAV-104 treatment at the highest tested concentration (p < 0.01) (Fig. 4A). We also tested the antiviral effect of PAV-104 on the emerging SARS-CoV-2 variants, Delta and Omicron, in AECs. Administration of PAV-104 also significantly reduced Delta and Omicron replication in primary AECs (p < 0.01) (Fig. 4B). Together, these data demonstrate that PAV-104 exerts potent antiviral activity against a broad range of circulating SARS-CoV-2 variants in primary AECs.

PAV-104 interferes with post-entry steps of the SARS-CoV-2 life cycle

Since PAV-104 was identified based on the inhibition of the viral particle assembly/budding process, we next sought to determine whether PAV-104 inhibits SARS-CoV-2 replication by acting on a post-entry step of the SARS-CoV-2 viral replication cycle as expected. We treated Calu-3 cells with PAV-104 at the concentration of 50 nM before or after virus infection. Protocols are illustrated in Fig. 5A. Pre-infection treatment with PAV-104 did not inhibit infectious virus release into culture supernatants (Fig. 5B), indicating that PAV-104 does not act on early steps in the SARS-CoV-2 life cycle (e.g. viral attachment and entry). Post-infection treatment with PAV-104 did strikingly reduce SARS-CoV-2 viral titer in the supernatant by measuring TCID₅₀ (p < 0.01), as compared to post-infection treatment with DMSO (negative control) or pre-infection treatment with PAV-104 (Fig. 5B). Consistent with these data, SARS-CoV-2 replication in primary AECs was decreased significantly by treatment with PAV-104 post viral infection (p < 0.05) (Fig. 5C, D). These results suggest that PAV-104 activity can be entirely attributed to blocking the late stage of the SARS-CoV-2 viral life cycle after viral entry.

PAV-104 blocks SARS-CoV-2 viral particle formation

Transient coexpression of four SARS-CoV-2 structural proteins (N, M, E, and S) in cell culture has been shown to produce assembling virus-like particles (VLPs), which can be used to study the viral life cycle such as assembly/budding, egress, and entry^29,30. To explore whether PAV-104 results in the inhibition of SARS-CoV-2 viral formation/budding, we quantified production of SARS-CoV-2 structural proteins in VLPs from cell culture supernatants of transfected HEK-293T cells treated with PAV-104 or DMSO. Viral assembly was quantified by western blot and nanoparticle tracking analysis (NTA) of extracellular vesicles and viral particles. Western blots were performed on proteins from the pellet after ultracentrifugation of transfected cell lysates and culture supernatants. PAV-104 significantly reduced structural protein production in the pellet collected from cell supernatants in a dose-dependent manner (p < 0.01), but did not inhibit structural protein synthesis and steady-state levels of actin in the cell lysates (Fig. 6A, B, C). Consistent with western blot data (Fig. 6A), our NTA results showed that cells transfected with the four SARS-CoV-2 structural proteins displayed increased nanoparticle production as compared to cells transfected with empty vectors (p < 0.001) (Fig. 6D), reflecting production and release of SARS-CoV-2 VLPs. PAV-104 treatment inhibited the concentration of nanoparticles in the supernatants of cells transfected with the four SARS-CoV-2 structural proteins in a dose-dependent manner (p < 0.01) (Fig. 6D), while no effect on nanoparticle secretion in empty vector-transfected cell supernatants was observed (suggesting that extracellular vesicle secretion is not affected by PAV-104). These data indicate that PAV-104 specifically inhibits virus-like particle production in our model, and blocks SARS-CoV-2 replication through targeting the viral assembly/budding process.

PAV-104 inhibits the oligomerization of the SARS-CoV-2 N

To investigate the main drug target of PAV-104 involved in the SARS-CoV-2 viral particle assembly/budding process, drug resin affinity chromatography (DRAC) was performed as described previously²³. PAV-104 was coupled to the 4% crosslinked agarose resin as previously described²³. Cellular extracts from Calu-3 cells with or without SARS-CoV-2 infection were incubated on the PAV-104 drug resin columns with or without PAV-104 covalently attached, allowing the target to bind. After washing, specifically bound material was eluted with free drug (PAV-104), followed by stripping of remaining bound material from the drug and control columns with 1% SDS. Based on western blotting with an anti-SARS-CoV-2 N antibody, negligible SARS-CoV-2 N was bound to or eluted from control columns to which infected lysates were applied, while abundant SARS-CoV-2 N was bound to and eluted from columns of PAV-104 attached to resin (Fig. 7A), indicating that SARS-CoV-2 N is a major component of the target multi-protein complex. No N reactivity was observed in columns loaded with uninfected lysates as expected. The oligomerization of the N of SARS-CoV-2 has been demonstrated to be responsible for helping virus envelope formation and particle assembly^31–33. To determine if PAV-104 affects the oligomerization of SARS-CoV-2 N to inhibit SARS-CoV-2 viral particle assembly, cells were transfected with N in the presence or absence of PAV-104, followed by analysis with glycerol gradient ultracentrifugation and a commercial ELISA kit to determine N concentrations in fractions. N-expressing cells treated with PAV-104 showed that there is significant reduction of the N intermediate complex (p < 0.01) (Fraction 20–22) as compared to DMSO treatment (Fig. 7B), indicating that the oligomerization of SARS-CoV-2 N is inhibited by PAV-104 treatment. These data support a model in which PAV-104 directly or indirectly affects the oligomerization of SARS-CoV-2 N to inhibit viral particle formation/assembly.

PAV-104 treatment inhibits SARS-CoV-2 RNA expression and reduces ‘maturation of nucleoprotein’

Finally, to understand the transcriptional impact of PAV-104 in the setting of SARS-CoV-2 infection and immunopathology, we performed RNA-seq analysis on ALI-cultured primary AECs from 5 different healthy donors infected for 36 hours with SARS-CoV-2 in the presence or absence of PAV-104. Uninfected, untreated cells (Control) were characterized as a negative control and reference for two experimental conditions: SARS-CoV-2 infection and SARS-CoV-2 infection with PAV-104 treatment. SARS-CoV-2 infection was also used as a reference for SARS-CoV-2 infection with PAV-104 treatment. Differentially-expressed gene (DEG) analysis showed 81 genes were significantly up-regulated by SARS-CoV-2 infection alone (Fig. 8A, Table S1), with most of them being interferon-related genes, such as OAS3, HELZ2, IFIT3, OAS1, and ISG15. SARS-CoV-2 infection in the presence of PAV-104 exhibited a dramatic impact on the host transcriptome when compared to uninfected control, with 10,255 DEGs identified (Fig. 8B, Table S2), including 5,843 down-regulated and 4,412 up-regulated genes. In addition, when compared with SARS-CoV-2 infection alone, SARS-CoV-2 infection in the presence of PAV-104 exhibited a distinct transcriptomic signature, with 9,319 DEGs identified (Fig. 8C, Table S3). GSEA pathway enrichment analysis for Reactome datasets revealed that the interferon (IFN) signaling pathway was the most up-regulated pathway by SARS-CoV-2 infection (Fig. 8D, Table S4). Virus-induced IFN signaling pathways were reversed by PAV-104 treatment. Although IFN signaling pathway play an important role in protecting the host from the spreading of SARS-CoV-2³⁴, the IFN signaling pathway has also been found to be critical in initiating deleterious lung inflammatory responses^35,36.

Of particular interest in our transcriptomic data is the set of genes related to the ‘maturation of nucleoprotein’ pathway that are selectively upregulated by SARS-CoV-2 infection but not by SARS-CoV-2 infection with PAV-104 treatment (Fig. 8D). SARS-CoV-2 nucleoprotein is found in the host cell cytosol, the nucleus and plasma membrane³⁷. The ‘maturation of nucleoprotein’ signaling pathway, including oligomerization, ADP-ribosylation, phosphorylation, sumoylation, methylation and other post-translational modifications of nucleoprotein, are responsible for N movement, interaction with genomic RNAs, interaction with other proteins, and viral particle assembly^{16, 38–40}. Therefore, our observation that PAV-104 suppresses the ‘maturation of nucleoprotein’ signaling pathway reinforces inhibition of viral assembly/budding as the proposed mechanism of action underlying PAV-104 anti-SARS-CoV-2 activity.

We also examined the impact of PAV-104 treatment on SARS-CoV-2 expression by aligning sequencing reads against the SARS-CoV-2 reference genome. The number of reads mapping to each region of the viral genome was calculated and interpreted to infer viral expression patterns (Fig. 8E)²⁸. Consistent with the antiviral effect of PAV-104, the transcription of SARS-CoV-2 was profoundly suppressed in the presence of PAV-104. These results confirmed the highly potent antiviral activity of PAV-104 against SARS-CoV-2.

The rapid emergence and spreading of the SARS-CoV-2 Omicron variant that evades many monoclonal antibody therapies illustrates the need for anti-viral treatments with low susceptibility to evolutionary escape. Capsid assembly is an essential step in the viral life cycle mediated by the interaction of viral capsid proteins. Inhibition of this process can be used as a therapeutic approach; any proteins, any modifications, or any interactions which participate in or stabilize viral particle assembly in the producer cell can be manipulated to inhibit assembly, prevent release, and protect as-yet-uninfected target cells from subsequent infection.

In our prior work, we identified three small molecules, PAV-431, PAV-471, and PAV-104, as inhibitors of influenza virus assembly using our cell-free protein synthesis and viral assembly screening system²³. We further demonstrated that PAV-104 in particular exerted highly potent antiviral effects against Nipah virus, respiratory syncytial virus, adenovirus, and human rhinovirus with minimal toxicity²³. Here, building on these observations, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 infection. Our results show that PAV-104 inhibits SARS-CoV-2 replication in airway epithelial cells, exhibiting potent antiviral effects against a broad spectrum of circulating viral variants.

We have established that PAV-104 interferes with a post-entry step of the SARS-CoV-2 life cycle and blocks SARS-CoV-2 viral particle assembly/budding based on the following observations: 1) the chemotype of PAV-104 investigated here has no effects on early viral life cycle events (e.g. viral entry), 2) PAV-104 reduces virus release into the cell culture supernatant, 3) PAV-104 treatment does not reduce steady-state levels of cellular proteins and does not impede the translation of viral structural proteins, and 4) PAV-104 interacts with SARS-CoV-2 N and interferes with its oligomerization. Dimerization and oligomerization of SARS-CoV-2 N proteins is essential to enable associations with viral genomic RNA and other viral structural proteins (M, E, and S), playing a critical role in virus particle assembly^33,41. In addition to viral particle assembly, the coronavirus N is required for viral mRNA and genome synthesis, viral core formation, and virus budding/envelope formation⁴². Based on our data revealing interaction between PAV-104 and SARS-CoV-2 N, PAV-104 treatment may also affect other key post-entry steps in the viral cycle beyond virus assembly.

Previously, we showed that PAV-104 bound a small subset of the known allosteric modulator 14-3-3, itself implicated in the interactome of SARS-CoV-2^23,43,44. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shutting and other functions of N⁴⁵. In addition, human 14-3-3 proteins were reported to bind the mutational hotspot region of SARS-CoV-2 N and modulate SARS-CoV-2 N phosphoregulation⁴⁶. In accordance with these observations, our transcriptomic data showed that PAV-104 treatment negatively regulates the ‘maturation of nucleoprotein’ signaling pathway of SARS-CoV-1/2. For example, sumoylation of SARS-CoV-2 N protein can enhance its interaction affinity with itself and is critical for its nuclear translocation, which is in turn critical for N-mediated viral RNA genome packaging and interaction with M protein^40,47. Phosphorylation of SARS-CoV-2 N protein was reported to be responsible for its localization, phase-phase separation and interaction with host factors³¹. The precise manner in which PAV-104 affects the post-translational modification of SARS-CoV-2 N warrants additional investigation, which may reveal novel antiviral mechanisms and pharmacological targets.

Our transcriptomic analysis also revealed that PAV-104 treatment of infected cells reduced the expression of specific IFN-regulated genes and reversed SARS-CoV-2 induction of the IFN signaling pathway. IFN signaling is critical to antiviral responses^34,48. To counteract host defense, multiple studies have demonstrated that SARS-CoV-2 uses a multitude of mechanisms to avoid type-I IFN-mediated immune responses⁴⁹. On the other hand, robust type I IFN responses have been associated with severe COVID-19 disease, and may exacerbate hyperinflammation during the development of severe COVID-19^50,51. Therefore, beyond inhibition of viral replication, PAV-104 may exert adjunctive anti-inflammatory effects via selective suppression of interferon pathway members that enhances its clinical potential as a therapeutic for COVID-19.

In summary, our findings demonstrate that PAV-104, a host-targeted pan-viral small molecule inhibitor, is a promising therapeutic candidate for SARS-CoV-2.

Cell lines

Human lung adenocarcinoma epithelial Calu-3 cells (ATCC-HTB-55) were cultured in Eagle’s Minimum Essential Medium (EMEM). African green monkey kidney Vero E6 cells (ATCC-CRL-1586) and human kidney HEK29T cells (ATCC-CRL-3216) were cultured in Dulbecco’s Modified Eagle's medium (DMEM). All media were supplemented with 10% FBS and 1% penicillin/streptomycin. Vero E6 cells stably expressing TMPRSS2 (Vero E6-TMPRSS2) were established and cultured in DMEM in the presence of puromycin (1µg/ml).

All cells had been previously tested for mycoplasma contamination and incubated at 37°C in a humidified atmosphere with 5% CO2.

Primary airway epithelial cells (AECs)

Primary AECs were obtained and cultured as previously described²⁸. Briefly, Human unused donor tracheobronchial tissue was obtained at the time of lung transplant. The tissue was washed and placed in DMEM with 0.1% protease and antibiotics overnight at 4ºC. The next day, the solution was agitated, and the remaining tissue was removed. Cell pellets were treated with 0.05% trypsin-EDTA, then filtered through a cell strainer. Cells were plated onto 6mm/0.4mm Transwell ALI insert after treatment with FNC coating mixture. 10% FBS in DMEM and ALI media were added in equal volumes to each basal compartment and cultures were incubated at 37ºC with 5% CO₂. The next day, the media was removed and both compartments were washed with PBS and antibiotics. ALI media was then added to each basal compartment and changed every three days for at least 28 days until differentiated airways were ready for use .

Viruses

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains USA-WA1/2022, lineage P.1, lineage B.1.617.2, and lineage B.1.1.529 were obtained from BEI Resources of the National Institute of Allergy and Infectious Diseases (NIAID) and propagated in Vero E6-TMPRSS2 cells. Virus titer was measured in Vero E6 cells by TCID₅₀ assay. All the studies involving live viruses were conducted in the Vitalant Research Institute BSL-3 under approved safety protocols.

Ethics Statement

The studies involving human participants were reviewed and approved by the Human Research Protection Program, University of California, San Francisco. The patients/participants provided their written informed consent to participate in this study.

Preparation of PAV-104

The synthetic method of PAV-104 was illustrated in Fig. 1. To a solution of aldehyde1 (10 g, 65.79 mmol, 1.0 eq) in toluene was added 2,4-dimethoxybenzyl amine 2 (10.99 g, 65.79 mmol, 1.0 eq) and the reaction mixture was heated at 80°C for 24 h. Solvent was removed and the residue was taken in MeOH and cooled using an ice bath. Then sodium borohydride (4.97g, 131.58 mmol, 2.0 eq) was added slowly and the reaction mixture was stirred at room temperature for 12 h. Solvent was removed and residue was taken in ethyl acetate and then sat. NaHCO3 was added and stirred for 1 h. Organic layer was separated, dried (MgSO4) and solvent was removed to give amine 3, which was used in the next step without further purification.

To a solution of the crude amine 3 (5.0 g, 19.1 mmol, 1.0 eq) in DMF (25 mL) were added acid 4 (3.17 g, 19.1 mmol, 1.0 eq), HATU (8.7 g, 22.92 mmol, 1.2 eq,), and DIEA (12.32 g, 95.5 mmol, 5.0 eq) and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was then diluted with ethyl acetate (EtOAc) and washed with 10% aqueous HCl (1X), sat. NaHCO3 (1X) and water (3X). Organic layer was collected, dried (MgSO4) and evaporated to give a residue, which was taken in MeOH and then K2CO3 (2.64 g. 19.1 mmol, 1.0 eq) was added and stirred at room temperature for 12 h. Solvent was removed and the residue was taken in Ethyl acetate and washed with 10% HCl (1X). Organic layer was separated, dried and solvent was removed to give a residue, which was purified by column chromatography (EtOAc/ Hexane) to give compound 5.

To a stirred solution of compound 5 (1.0 g, 2.22 mmol, 1.0 eq) and cesium carbonate (1.08 g, 3.33 mmol, 1.5 eq) in DMF (15 mL) was added methyl 4-(chloromethyl) benzoate 6 (450 mg, 2.44 mmol, 1.2 eq) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ethyl acetate and washed with water (3x). Organic layer was dried and concentrated to give crude product 7. The crude compound 7 was stirred in a 1:1 mixture of TFA: DCM for 12 h. Concentration followed by chromatography purification (Hexane/ EtOAc) provided compound 8.

To a stirred solution of compound 8 (0.84 mmol, 1.0 eq) in 3:1 mixture of THF: H₂O (12 mL) was added LiOH (40 mg, 1.68 mmol, 2.0 eq) and the reaction mixture was stirred at 65°C for 12 h. The reaction mixture was evaporated under vacuum to give a residue, which was stirred in a mixture of 10% aqueous HCl and ethyl acetate for 30 min. Organic layer was collected, washed (H₂O, 1X), dried and concentrated to give crude acid 9.

To a solution of the amine 10 (68 mg, 0.552 mmol, 1.2 eq) in DMF (25 mL) were added acid 9 (200 mg, 0.46 mmol, 1.0 eq), HATU (210 mg, 0.552 mmol, 1.2 eq,) and DIEA (0.300 mg, 2.3 mmol, 5.0 eq). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was then diluted with EtOAc and washed with 10% aqueous HCl (1X), sat. NaHCO3 (1X) and water (3X). Organic layer was collected, dried (MgSO4) and evaporated to give a residue, which was purified by column chromatography (EtOAc/ Hexane) to give PAV-104.

Drug cytotoxicity assay

The cytotoxic effect of PAV-104 on Calu-3 cells was measured using an MTT assay kit (Abcam, ab211091) following the manufacturer’s instructions. In brief, Calu-3 cells were seeded in 96-well cell culture plates. Appropriate concentrations of PAV-104 were added to the medium (0-5000 nM). After 48 hours, the media was removed and 100 µl MTT reagent (1:1 dilution in DMEM medium (serum free)) was added to each well and incubated for 3 h at 37ºC. Then the medium was removed, and 150 µl MTT solvent was added into each well. Quantification was performed by reading absorbance at OD = 590 nm. The data from three independent experiments was used to calculate the CC₅₀ by nonlinear regression using GraphPad Prism 8.0 software.

SARS-CoV-2 infection and drug administration

Calu-3 cells were seeded at 0.5 x 10⁶ cells per well in 0.5 ml volumes using a 24-well plate, or were seeded at 1 x 10⁵ cells per well in 0.1 ml volumes using a 96-well plate. The following day, cells were pretreated with or without PAV-104 or remdesivir for one hour. Then viral inoculum (MOI of 0.01; 500 µl/well or 100µl/well) was prepared using EMEM containing indicated concentrations of PAV-104 or remdesivir and added to the wells. The inoculated plates were incubated at 37ºC with 5% CO₂. At indicated infection time points, supernatants were collected and stored at -80ºC. Cells were lysed with TRizol (Thermo Fisher Scientific, 15596026) for RNA extraction.

For infection of primary AECs in ALI culture, cells were pretreated with PAV-104 in the basal compartment for one hour. SARS-CoV-2 (diluted in ALI-culture medium, MOI = 0.1) was added to the apical chamber of inserts (250 µl) and the basal compartment (500 µl). Then the cultures were incubated for 2 hours at 37ºC (5% CO₂) to enable virus entry. Subsequently, the cells were washed and fresh ALI medium (500 µl) containing PAV-104 was added into the basal compartment. Cells were incubated at 37ºC (5% CO₂) and harvested for analysis at 36 hours post-infection.

Viral titer by TCID assay

Virus production in the supernatant was measured by quantifying TCID₅₀. Vero E6 cells were plated in 96-well plates at 5 X 10⁴ cells per well. The next day, supernatants collected from Calu-3 cells were subjected to 10-fold serial dilutions (10¹ to 10¹¹) and inoculated onto Vero E6 cells. The cells were incubated at 37ºC with 5% CO₂. Three to five days post infection, each inoculated well was evaluated for presence or absence of viral CPE. TCID₅₀ was calculated based on the method of Reed and Muench.

RT-qPCR

Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622) in accordance with the manufacturer's instructions. RT-qPCR was performed for each sample using Taqman Universal Master mix II, with UNG (Thermo Fisher Scientific, 4440038) on a ViiA7 Real time PCR system. Primers and probes for detection of the RNaseP gene and SARS-CoV-2 nucleocapsid (N) gene were obtained from IDT (2019-nCoV RUO Kit (Integrated DNA Technologies, 10006713)). The expression level of the N gene was determined relative to the endogenous control of the cellular RNaseP gene.

RNA-sequencing analysis

RNA concentration and quality was measured using High Sensitivity RNA ScreenTape Analysis (Agilent, 5067 − 1500). cDNA libraries were constructed and sequencing was performed by Novogene using their mRNA sequencing protocol. The raw RNA sequencing data were aligned to the human genome (GRCh38) using STAR (version 2.7.3a). Analysis of differential expression was performed using DESeq2 according to a standard protocol. Genes with adjusted P-value < 0.05 were considered as significantly differentially expressed. Gene set enrichment analysis was performed using the fgsea package (version 1.22.0) in R. The Reactome database (version 7.5.1) was downloaded from MSigDB (https://www.gsea-msigdb.org).

Immunofluorescence microscopy and image analysis

Cells were fixed and permeabilized with cold methanol : acetone (1:1) for 10 min at 4℃ according to our previous method²⁸. In brief, cells were then incubated with blocking buffer (5% goat serum (Seracare Life Sciences Inc, 55600007)), a primary antibody (monoclonal rabbit anti-SARS-CoV-2 N antibody (GeneTex, GTX135357)), a secondary antibody (Goat anti-Rabbit IgG (H + L) secondary antibody, FITC (Thermo Fisher, 65-6111)), and DAPI (Thermo Fisher Scientific, D1306). Images were acquired using a fluorescence microscope.

To measure the frequency of infected cells, randomly-selected areas were imaged. Each treatment had three replicates. The FITC-positive cells and DAPI-positive cells were quantified using CellProfiler software as previously described. The same threshold value was applied to the images of each area.

Quantification of the western blots was carried out with Image J software.

Production of SARS-CoV-2 virus-like particles (VLPs)

HEK293T cells were seeded in T75 cell culture flasks. The next day, cells were transfected with empty pcDNA3.1 plasmid or pcDNA3.1 plasmid encoding the SARS-CoV-2 M (Addgene-158078), E (Addgene-158080), N (Addgene-158079), and S proteins (Addgene-158074), as indicated. 1 µg of each plasmid was used, with 5 µg of total plasmid in each transfection, normalized using empty vectors, in 400 µl Opti-MEM and 18 µl of PEI. The transfection mixture was incubated at room temperature for 15 min and dropped into the HEK293T cells. Six hours post transfection, the media was removed and supplemented with fresh medium containing PAV-104 at indicated concentrations. The supernatant and cell lysate were collected after 60 hours. For the purification of SARS-CoV-2 VLPs, the supernatant was passed through a 0.45 µm syringe filter, then loaded on top of a 20% sucrose cushion in PBS, and ultracentrifuged at 30,000 rpm in an SW41 rotor for two hours. VLP-containing pellets were washed with ice cold PBS and resuspended in SDS loading buffer, followed by sonication in an ice-water bath. Or VLP-containing pellets were resuspended in PBS (passed through 0.22 µm syringe filter) for quantification by NTA. Cells were lysed in RIPA buffer (Thermo Fisher Scientific, 89900) and sonicated in an ice-water bath.

Immunoblots of SARS-CoV-2 VLPs

Total protein in pellet and cell lysate samples were separated by SDS-PAGE, and subsequently electro-transferred onto a supported PVDF membrane. Membranes were cut and probed for M with rabbit anti-SARS-CoV-2 M (Thermo Fisher Scientific, PA1-41160), N with rabbit anti-SARS-CoV-2 N (Rockland Immunochemicals, 200-401-A50), E with rabbit anti-SARS-CoV-2 E (Thermo Fisher Scientific, PA5-112047), and S with rabbit anti-SARS-CoV-2 S1/S2 (Thermo Fisher Scientific, PA5-112048) or with mouse anti-SARS-CoV-2 spike (Genetex Inc, GTX632604). Goat anti-rabbit IgG HRP and goat anti-mouse IgG HRP secondary antibodies were used as appropriate. β-actin was used as a cell lysate and pellet loading control by probing membranes with rabbit anti-human β-actin, conjugated with HRP (Cell Signaling Technology, 12620). All antibodies were diluted in 5% milk and membranes were washed with Tween 20 washing buffer (Thermo Fisher Scientific, J60304.K3). Chemiluminescent signal was visualized using SuperSignal West Femo Substrate (Thermo Fisher Scientific, PI34094) or using ECL Blotting Reagents (SIGMA, GERPN2109), and imaged using ImageQuant LAS 4000.

Quantification of VLPs by nanoparticle tracking analysis (NTA)

VLP-containing pellets were diluted in PBS (passed through 0.22 µm syringe filter) to a concentration in the range of 10⁷-10⁹/ml and examined using a NanoSight NS300 (NanoSight Ltd) equipped with a 405 nm laser. Five 60 s-long videos were taken for each sample with camera level 16 and the detection threshold set at 5. Raw data of particle movement and laser scattering were analyzed using NTA software (version 3.3, NanoSight Ltd). The output data were presented as nanoparticle concentration and size.

Drug Resin Affinity Chromatography (DRAC)

Drug Resin Affinity Chromatography experiments were performed where 30 µl of extract prepared from Calu3 cells under different infection and treatment conditions were adjusted to a protein concentration of approximately 2.3 mg/ml in column buffer and supplemented with an “energy cocktail” (to a final concentration of 1mM rATP, 1mM rGTP, 1mM rCTP, 1mM UTP, 4mM creatine phosphate, pH 7.6) and 5 µg/mL creatine kinase) and incubated on a column containing 30 µl of affi-gel resin coupled to either PAV-104 or a 4% agarose matrix (control) for one hour at room temperature. The PAV-104 resin conditions were run side-by-side in triplicate, while the control resin conditions were done in single point. The flow-through material was collected, and the resin was washed with 1.5 mL column buffer then eluted with 100 µl PAV-104 plus the energy cocktail at room temperature for two hours then stripped with 100 µl 1% SDS. The eluate and SDS-stripped material run on agarose gels and are analyzed by western blot for SARS-CoV-2 N protein (Rockland Immunochemicals, 200-401-A50).

Glycerol gradient sedimentation and ELISA-based assessment of SARS-CoV-2 N

Cell extracts from SARS-CoV-2 N-transfected HEK-293T cells in the presence or absence of PAV-104 were centrifuged at 15000 g for 10 mins at 4℃ to obtain the supernatant. 200 µl of supernatant were loaded on the top of a 5 ml continuous 10–40% glycerol gradient in lysis buffer (v/v, Pierce IP Lysis Buffer (Thermo Fisher, 87787)) prepared using the Gradient Master machine (Biocomp, Gradient Station). After concentration at 135000 g for 20 h at 4℃ in a SW55 rotor (Beckman Coulter), 22 fractions of 250 µl were collected from the top to the bottom of the gradient.

Proteins were assessed by the commercial SARS-CoV-2 N protein sandwich ELISA kit (GeneTex, GTX535824) following manufacturer's instructions. In brief, each fraction was diluted to 1:1000 using assay dilute reagent. 50 µl of each standard and samples were added into the appropriate wells, then incubated at room temperature for 2 hours. The solutions in the wells were aspirated and wells were washed with a washing buffer six times. Then the conjugate solution was added and incubated at room temperature for 1 hour. The solutions in the wells were aspirated and wells were washed with a washing buffer six times once again. TMB solution was added to the wells and incubated in darkness for 15 mins at room temperature. Stop solution was added to each well. Finally, optical density at 450 nm was read within 15 mins.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 8 software. Data were presented as means ± SEM or median. Data were analyzed for statistical significance using an unpaired or paired Student’s t test to compare two groups, or using a paired t test. Only p values of 0.05 or lower were considered statistically significant (p > 0.05 [ns], p ≤ 0.05 [*], p ≤ 0.01 [**], p ≤ 0.001 [***], p ≤ 0.0001 [****]).

Hu, B., Guo, H., Zhou, P. & Shi, Z.-L. Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol 19, 141–154 (2021).
Razonable, R. R. et al. Casirivimab–Imdevimab treatment is associated with reduced rates of hospitalization among high-risk patients with mild to moderate coronavirus disease-19. eClinicalMedicine 40, (2021).
Thilagar, B. P. et al. Anti-Spike Monoclonal Antibody Therapy in Pregnant Women With Mild-to-Moderate Coronavirus Disease 2019 (COVID-19). Obstet Gynecol 139, 616–618 (2022).
Chen, R. E. et al. In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains. Nature 596, 103–108 (2021).
Beigel, J. H. et al. Remdesivir for the Treatment of Covid-19 - Final Report. N Engl J Med 383, 1813–1826 (2020).
Jayk Bernal, A. et al. Molnupiravir for Oral Treatment of Covid-19 in Nonhospitalized Patients. N Engl J Med 386, 509–520 (2022).
Owen, D. R. et al. An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586–1593 (2021).
Wang, Y. et al. Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet 395, 1569–1578 (2020).
Kabinger, F. et al. Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nat Struct Mol Biol 28, 740–746 (2021).
Wang, L. et al. COVID-19 rebound after Paxlovid and Molnupiravir during January-June 2022. medRxiv 2022.06.21.22276724 (2022) doi:10.1101/2022.06.21.22276724.
Hashimoto, R. et al. Dual inhibition of TMPRSS2 and Cathepsin B prevents SARS-CoV-2 infection in iPS cells. Molecular Therapy - Nucleic Acids 26, 1107–1114 (2021).
Zhang, Q. et al. Molecular mechanism of interaction between SARS-CoV-2 and host cells and interventional therapy. Sig Transduct Target Ther 6, 1–19 (2021).
Mengist, H. M., Dilnessa, T. & Jin, T. Structural Basis of Potential Inhibitors Targeting SARS-CoV-2 Main Protease. Frontiers in Chemistry 9, (2021).
Narayanan, A. et al. Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay. Commun Biol 5, 169 (2022).
Ahamad, S., Gupta, D. & Kumar, V. Targeting SARS-CoV-2 nucleocapsid oligomerization: Insights from molecular docking and molecular dynamics simulations. J Biomol Struct Dyn 40, 2430–2443 (2022).
Cai, T., Yu, Z., Wang, Z., Liang, C. & Richard, S. Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication. J Biol Chem 297, 100821 (2021).
Bhowmik, D. et al. Identification of potential inhibitors against SARS-CoV-2 by targeting proteins responsible for envelope formation and virion assembly using docking based virtual screening, and pharmacokinetics approaches. Infect Genet Evol 84, 104451 (2020).
He, R. et al. Characterization of protein–protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus. Virus Research 105, 121–125 (2004).
Khambhati, K. et al. Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems. Frontiers in Bioengineering and Biotechnology 7, (2019).
Rodríguez-Limas, W. A., Sekar, K. & Tyo, K. E. J. Virus-like particles: the future of microbial factories and cell-free systems as platforms for vaccine development. Curr Opin Biotechnol 24, 1089–1093 (2013).
Spice, A. J., Aw, R., Bracewell, D. G. & Polizzi, K. M. Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System. Frontiers in Bioengineering and Biotechnology 8, (2020).
Lingappa, U. F. et al. Host–rabies virus protein–protein interactions as druggable antiviral targets. Proceedings of the National Academy of Sciences 110, E861–E868 (2013).
Müller-Schiffmann, A. et al. A Pan-respiratory Antiviral Chemotype Targeting a Transient Host Multiprotein Complex. 2021.01.17.426875 Preprint at https://doi.org/10.1101/2021.01.17.426875 (2022).
Reed, J. C. et al. Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly. J Virol 95, e00883-20 (2021).
Grein, J. et al. Compassionate Use of Remdesivir for Patients with Severe Covid-19. N Engl J Med 382, 2327–2336 (2020).
Pruijssers, A. J. et al. Remdesivir Inhibits SARS-CoV-2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA Polymerase in Mice. Cell Rep 32, 107940 (2020).
Ryu, G. & Shin, H.-W. SARS-CoV-2 Infection of Airway Epithelial Cells. Immune Netw 21, e3 (2021).
Du, L. et al. Human Galectin-9 Potently Enhances SARS-CoV-2 Replication and Inflammation in Airway Epithelial Cells. 2022.03.18.484956 Preprint at https://doi.org/10.1101/2022.03.18.484956 (2022).
Plescia, C. B. et al. SARS-CoV-2 viral budding and entry can be modeled using BSL-2 level virus-like particles. J Biol Chem 296, 100103 (2021).
Yurkovetskiy, L. et al. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. Cell 183, 739–751.e8 (2020).
Lu, S. et al. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein. Nat Commun 12, 502 (2021).
Cong, Y., Kriegenburg, F., de Haan, C. A. M. & Reggiori, F. Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers. Sci Rep 7, 5740 (2017).
Wu, C. et al. Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain. bioRxiv 2020.11.30.404905 (2020) doi:10.1101/2020.11.30.404905.
Diamond, M. S. & Kanneganti, T.-D. Innate immunity: the first line of defense against SARS-CoV-2. Nat Immunol 23, 165–176 (2022).
Makris, S., Paulsen, M. & Johansson, C. Type I Interferons as Regulators of Lung Inflammation. Front Immunol 8, 259 (2017).
Sodeifian, F., Nikfarjam, M., Kian, N., Mohamed, K. & Rezaei, N. The role of type I interferon in the treatment of COVID-19. J Med Virol 94, 63–81 (2022).
Scherer, K. M. et al. SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress. Sci Adv 8, eabl4895 (2022).
Cubuk, J. et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat Commun 12, 1936 (2021).
Grunewald, M. E., Fehr, A. R., Athmer, J. & Perlman, S. The coronavirus nucleocapsid protein is ADP-ribosylated. Virology 517, 62–68 (2018).
Li, F. Q., Xiao, H., Tam, J. P. & Liu, D. X. Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus. FEBS Lett 579, 2387–2396 (2005).
Ye, Q., West, A. M. V., Silletti, S. & Corbett, K. D. Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein. Protein Sci 29, 1890–1901 (2020).
McBride, R., van Zyl, M. & Fielding, B. C. The coronavirus nucleocapsid is a multifunctional protein. Viruses 6, 2991–3018 (2014).
Gordon, D. E. et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583, 459–468 (2020).
Zhou, Y. et al. A comprehensive SARS-CoV-2–human protein–protein interactome reveals COVID-19 pathobiology and potential host therapeutic targets. Nat Biotechnol 41, 128–139 (2023).
Surjit, M. et al. The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation. J Virol 79, 11476–11486 (2005).
Tugaeva, K. V. et al. Human 14-3-3 Proteins Site-selectively Bind the Mutational Hotspot Region of SARS-CoV-2 Nucleoprotein Modulating its Phosphoregulation. J Mol Biol 435, 167891 (2023).
Madahar, V., Rodgers, V. G. J. & Liao, J. SUMOylation of SARS-CoV-2 Nucleocapsid protein enhances its interaction affinity and plays a critical role for its nuclear translocation. 2022.05.31.494262 Preprint at https://doi.org/10.1101/2022.05.31.494262 (2022).
Kim, Y.-M. & Shin, E.-C. Type I and III interferon responses in SARS-CoV-2 infection. Exp Mol Med 53, 750–760 (2021).
Gu, W. et al. The molecular mechanism of SARS-CoV-2 evading host antiviral innate immunity. Virol J 19, 49 (2022).
Akamatsu, M. A., de Castro, J. T., Takano, C. Y. & Ho, P. L. Off balance: Interferons in COVID-19 lung infections. EBioMedicine 73, 103642 (2021).
Hadjadj, J. et al. Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369, 718–724 (2020).

Acknowledgement

This study was supported by The Sandler Program for Breakthrough Biomedical Research (UCSF PBBR) and NIH grant R01MH112457 (to SKP); Veterans Affairs Office of Research and Development CX002011 and NIH R01HL151552 (to JRG).

Author contributions

L.D. and S.P. initiated the project and designed the experiments. L.D., F.D., and J.G. performed and processed RNA-seq. F.D. and J.G. isolated and prepared ALI-cultured primary airway epithelial cells (AECs). L.D., M.B., and G.S. performed MTT and glycerol gradient sedimentation assay. J.B. analyzed RNA-seq data. L.D. and P.D. quantified VLPs by NTA. G.S. performed SARS-CoV-2 stock. S.S., A.L., M.M., S.Y., K.P., V.L., and H.B. prepared PAV-104 and performed the DRAC assay. L.D., V.L., and S.P. prepared the manuscript. J.G. and S.P. jointly supervised the work.

Competing interests

Suganya Selvarajah, Anuradha F. Lingappa, Maya Michon , Shao Feng Yu, and Kumar Paulvannan are employees of Prosetta Biosciences. Vishwanath R. Lingappa is the CEO of Prosetta Biosciences, which manufactures PAV-104 presented in the manuscript. The authors declare that there are no other competing interests.

Yes there is potential Competing Interest. Suganya Selvarajah, Anuradha F. Lingappa, Maya Michon , Shao Feng Yu, and Kumar Paulvannan are employees of Prosetta Biosciences. Vishwanath R. Lingappa is the CEO of Prosetta Biosciences, which manufactures PAV-104 presented in the manuscript. The authors declare that there are no other competing interests.

TableS1.xlsx
Dataset 1
TableS2.xlsx
Dataset 2
TableS3.xlsx
Dataset 3
TableS4.xlsx
Dataset 4

Download PDF

Journal Publication

published 22 Apr, 2024

Read the published version in Communications Biology →

Version 1

posted

You are reading this latest preprint version

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

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

PAV-104 suppresses SARS-CoV-2 infection in Calu-3 cells

PAV-104 and remdesivir potently inhibit SARS-CoV-2 replication

PAV-104 is a highly potent antiviral inhibitor of SARS-CoV-2 in primary airway epithelial cells

PAV-104 interferes with post-entry steps of the SARS-CoV-2 life cycle

PAV-104 blocks SARS-CoV-2 viral particle formation

PAV-104 inhibits the oligomerization of the SARS-CoV-2 N

PAV-104 treatment inhibits SARS-CoV-2 RNA expression and reduces ‘maturation of nucleoprotein’

Discussion

Methods

Cell lines

Primary airway epithelial cells (AECs)

Viruses

Ethics Statement

Preparation of PAV-104

Drug cytotoxicity assay

SARS-CoV-2 infection and drug administration

Viral titer by TCID assay

RT-qPCR

RNA-sequencing analysis

Immunofluorescence microscopy and image analysis

Production of SARS-CoV-2 virus-like particles (VLPs)

Immunoblots of SARS-CoV-2 VLPs

Quantification of VLPs by nanoparticle tracking analysis (NTA)

Drug Resin Affinity Chromatography (DRAC)

Glycerol gradient sedimentation and ELISA-based assessment of SARS-CoV-2 N

Statistical analysis

References

Declarations

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1