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

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.


Introduction
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, belongs to a highly contagious betacoronavirus 1 . 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 e cacy 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 e cacy of anti-SARS-CoV-2 mAbs is negligible in the face of currently circulating viral variants 4 . Beyond mAbs, antiviral small-molecule drugs have been developed that target speci c 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][5][6] and a viral 3C-like protease inhibitor, paxlovid, which consists of nirmatrelvir and ritonavir 7 . Clinical studies have shown that remdesivir is not associated with statistically signi cant clinical bene ts 8 . In vitro studies have shown that molnupiravir is used as a substrate by host RNA polymerases including the mitochondrial DNA-dependent RNA polymerase 9 . Paxlovid treatment is often associated with COVID-19 rebound following the treatment cycle 10 . Taken together, these realities illustrate the need for effective and broad-spectrum antiviral drugs for COVID-19 with minimal off-target effects. 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 signi cantly 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 signi cantly by PAV-104 in a dose dependent manner, as measured by median tissue culture infectious dose (TCID 50 ) (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 con rmed by speci c staining of viral N using an immuno uorescence 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 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 rst gateway for SARS-CoV-2 infection 27 . 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 vivo 28 . 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 signi cantly 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 identi ed 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 TCID 50 (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 signi cantly 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 quanti ed 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 quanti ed 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 signi cantly 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), re ecting 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 speci cally 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 a nity chromatography (DRAC) was performed as described previously 23 . PAV-104 was coupled to the 4% crosslinked agarose resin as previously described 23 . 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, speci cally 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][32][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 signi cant 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.  Table S3). GSEA pathway enrichment analysis for Reactome datasets revealed that the interferon (IFN) signaling pathway was the most upregulated 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-2 34 , the IFN signaling pathway has also been found to be critical in initiating deleterious lung in ammatory 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 membrane 37 . The 'maturation of nucleoprotein' signaling pathway, including oligomerization, ADP-ribosylation, phosphorylation, sumoylation, methylation and other post-translational modi cations 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 con rmed the highly potent antiviral activity of PAV-104 against SARS-CoV-2.

Discussion
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 modi cations, 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 identi ed three small molecules, PAV-431, PAV-471, and PAV-104, as inhibitors of in uenza virus assembly using our cell-free protein synthesis and viral assembly screening system 23 . 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 toxicity 23 . 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 formation 42 . 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 N 45 . 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 phosphoregulation 46 . 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 a nity 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 factors 31 . The precise manner in which PAV-104 affects the posttranslational modi cation 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 speci c 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 responses 49 . On the other hand, robust type I IFN responses have been associated with severe COVID-19 disease, and may exacerbate hyperin ammation during the development of severe COVID-19 50,51 .
Therefore, beyond inhibition of viral replication, PAV-104 may exert adjunctive anti-in ammatory effects via selective suppression of interferon pathway members that enhances its clinical potential as a therapeutic for COVID-19.
In summary, our ndings demonstrate that PAV-104, a host-targeted pan-viral small molecule inhibitor, is a promising therapeutic candidate for SARS-CoV-2.
All cells had been previously tested for mycoplasma contamination and incubated at 37°C in a humidi ed atmosphere with 5% CO2.

Primary airway epithelial cells (AECs)
Primary AECs were obtained and cultured as previously described 28 . Brie y, 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 ltered 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% CO 2 . 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 .

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 puri cation.
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 puri ed 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 puri cation (Hexane/ EtOAc) provided compound 8.
To a stirred solution of compound 8 (0.84 mmol, 1.0 eq) in 3:1 mixture of THF: H 2 O (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 (H 2 O, 1X), dried and concentrated to give crude acid 9.

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. Quanti cation was performed by reading absorbance at OD = 590 nm. The data from three independent experiments was used to calculate the CC 50 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 10 6 cells per well in 0.5 ml volumes using a 24-well plate, or were seeded at 1 x 10 5 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% CO 2 . At indicated infection time points, supernatants were collected and stored at -80ºC. Cells were lysed with TRizol (Thermo Fisher Scienti c, 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% CO 2 ) 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% CO 2 ) and harvested for analysis at 36 hours post-infection.

Viral titer by TCID assay
Virus production in the supernatant was measured by quantifying TCID 50 . Vero E6 cells were plated in 96well plates at 5 X 10 4 cells per well. The next day, supernatants collected from Calu-3 cells were subjected to 10-fold serial dilutions (10 1 to 10 11 ) and inoculated onto Vero E6 cells. The cells were incubated at 37ºC with 5% CO 2 . Three to ve days post infection, each inoculated well was evaluated for presence or absence of viral CPE. TCID 50 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 Scienti c, 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 Scienti c, 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 signi cantly 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).
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 quanti ed using CellPro ler software as previously described. The same threshold value was applied to the images of each area.
Quanti cation 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 asks. 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 puri cation of SARS-CoV-2 VLPs, the supernatant was passed through a 0.45 µm syringe lter, 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 lter) for quanti cation by NTA. Cells were lysed in RIPA buffer (Thermo Fisher Scienti c, 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- Quanti cation of VLPs by nanoparticle tracking analysis (NTA) VLP-containing pellets were diluted in PBS (passed through 0.22 µm syringe lter) to a concentration in the range of 10 7 -10 9 /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 A nity Chromatography (DRAC)
Drug Resin A nity 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 nal 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 a -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 owthrough 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). 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 signi cance using an unpaired or paired Student's t test to compare two groups, or using a paired t test.