Restriction of SARS-CoV-2 replication by receptor transporter protein 4 (RTP4)

To study host factors that restrict the replication of beta-CoVs, we tested two family members: the pandemic COVID-19 virus SARS-CoV-2 and the seasonal cold virus HCoV-OC43. For these studies, we tested several cell lines (CHME3, 293T, Hela, MRC5, BHK21, A549, Huh7, and Vero) to identify one that would support the replication of both viruses. Of the cell lines tested, only MRC5 and the human microglial cell-line CHME3 supported HCoV-OC43 (not shown). However, neither expressed the SARS-CoV-2 receptor ACE2 or supported SARS-CoV-2 replication. In light of the favorable growth characteristics of CHME3, we established a clonal CHME3 cell line, ACE2.CHME3, by lentiviral vector transduction that expressed high levels of ACE2 (Fig. S1). To further evaluate the ability of CHME3 cells to support HCoV-OC43 replication, we infected the cells at a multiplicity of infection (MOI) of 0.5 and over the next 72 h, quantified the viral genomic and subgenomic RNA. Genomic viral RNA was first detected 12 h post-infection and increased until 72 h after which the cells died. Similar results were obtained for the subgenomic RNA which is a measure of active virus replication (Fig. 1A). To test the ability of the cells to support SARS-CoV-2 replication, CHME3 and ACE2.CHME3 cells were infected with SARS-CoV-2 WA1/2020 at an MOI of 0.1 and quantified the genomic and subgenomic RNAs. CHME3 cells did not support virus replication, while in the ACE2.CHME3 cells, viral genomic and subgenomic RNAs rose to high levels at 1 day post-infection (dpi) and increased another 10-fold 3 dpi (Fig. 1B).

To identify host factors that restrict CoV replication, we generated a list of genes induced by type-I IFN in primary human monocytes. Monocytes were chosen because they are highly sensitive to type-I IFN and might induce genes that are not typically induced by type-I IFN in transformed cell lines. Monocytes from three donors were treated for 24 h with type-I IFN or were mock treated. RNA was then prepared and subjected to analysis by RNA-seq (47). In order to analyze antiviral activity of ISGs, 14 genes with log₂-fold change (log2FC) >5 were selected, most of which had previous associations with antiviral activity and encompassed ISGs with previously characterized broad-acting antiviral activities (Fig. 1C; Table S1) (7, 19, 48). We established stable CHME3 cell lines by lentiviral vector transduction for these (RTP4, IFI44L, ISG15, ISG20, MX1, TRIM22, EIF2AK2, IFIT1, IFIT2, IFIT3, IFIT5, GBP1, GBP5, and viperin) (47). To test for restriction of CoV replication by the candidate genes, we infected the CHME3 stable cell lines with HCoV-OC43 and analyzed virus replication 2 dpi by indirect immunofluorescence for the viral N protein using high-content microscope imaging. The results showed that the cell line expressing hRTP4 restricted the replication of the virus (Fig. 1D), while the other 13 genes had no effect on virus replication. The hRTP4 expressing CHME3 cells were not completely protected as 30% of the cells remained infected; however, as the cell lines are polyclonal, they express the proteins at heterogeneous levels which would allow for the infection of some of the cells. Small effects were noted for viperin, ISG15, and IFIT3 but these were not statistically significant.

Our RNA-seq analysis showed that hRTP4 is IFN-inducible in human cells (47). Recent studies have shown SARS-CoV-2 infection of macrophages in vivo and ex vivo (49 - 51). To determine whether CoV infection would induce RTP4 expression in mice, we infected K1 mice that express human ACE2 from a keratin promoter and K18 mice that express ACE2 from the endogenous promoter with WA1/2020 SARS-CoV-2 and measured the relative abundance of murine RTP4 (mRTP4) and several ISG mRNA transcripts in the lungs. The results showed that mRTP4 mRNA transcripts were strongly induced by SARS-CoV-2 infection in K1-hACE2 and K18-hACE2 transgenic mice (Fig. 1E). The gene was induced more strongly in K18 mice (about 100-fold) a level that was similar to highly inducible mMX1. The induction was about 10-fold in K1 mice which support lower levels of virus replication. mRTP4 was induced to a level greater than those of well-known ISGs mOAS1, mISG15, and mIFIT3. The mRTP4 mRNA copy number in the infected mouse lung cells was comparable to that in the CHME3.hRTP4 cell line, suggesting that the cell line expressed a physiological level of hRTP4 mRNA transcript (Fig. 1F).

To determine the potency of hRTP4 antiviral activity, we transfected CHME3 cells with a range (0.25–4 µg) of 2X-FLAG-tagged hRTP4 (hRTP4-2F) expression vector or with (0.25–4 µg) of empty vector. Relative hRTP4 protein levels in the transfected cells were determined on an immunoblot probed with anti-FLAG antibody (Fig. 2A). The transfected CHME3 cells were then infected with HCoV-OC43 and virus replication was measured 48 h post-infection by immunoblot analysis of cell lysates for the HCoV-OC43 N protein and by quantification of the viral genomic and subgenomic RNAs by qRT-PCR. The analysis showed a 70% reduction in N protein with 4 and 2 µg pLenti.hRTP4 plasmid and decreased inhibition with lesser amounts of plasmid (Fig. 2A, lower panel) with the band intensities shown in the histogram (Fig. 2A, right). To determine how the level of protein expression in the transfected CHME3 cells compared to those in primary cells expressing physiological levels of RTP4, we analyzed the protein in type-I IFN-treated CHME3, peripheral blood mononuclear cells (PBMCs) and THP-1 cells. The results confirmed the IFN-inducibility of RTP4 in the three cell types and showed that the levels of protein were comparable to those in transfected cells that were sufficient to inhibit virus replication (Fig. 2B).

Analysis of the viral genomic and subgenomic RNA levels showed that 4 µg of hRTP4 plasmid decreased the genomic RNA by about 1,000-fold with a similar decrease in subgenomic viral RNA. As little as 0.5 µg of transfected expression vector plasmid causes a significant decrease in viral RNA (Fig. 2C). To determine whether hRTP4 was active against SARS-CoV-2 variants, we infected the ACE2.CHME3 cells at 0.5 and 0.05 MOI with Omicron variants BA.1 and BA.2 and quantified viral RNA 2 dpi. The results showed that hRTP4 resulted in a >100,000-fold reduction in genomic BA.1 viral RNA at MOI of 0.05 and about 10,000-fold reduction at MOI of 0.5 (Fig. 2D). BA.2 did not replicate to as high titer but the impact of hRTP4 was evident. The effect was even more pronounced for subgenomic RNA, probably because of the absence of input viral RNA copies, unlike for the analysis of the genomic RNA for which input viral RNA is present prior to virus replication.

The CoV replication cycle is initiated by the synthesis of negative-strand RNA using the incoming positive-strand genomic RNA as template. RNA synthesis occurs in the cytoplasm on double-membrane vesicle replication organelles (ROs) that serve as the recruitment site for viral replicative proteins and specific host factors (52). This strategy results in the accumulation of dsRNA and viral N protein as a replicative intermediate within the cytoplasmic viral ROs. The N protein containing ROs in HCoV-OC43-infected cells can be detected with specific antibody. N protein accumulation can be used as a measure of the recruitment of replication complexes that in-turn are related to virus replication initiation. To determine the step in the CoV life cycle that is restricted by hRTP4, HCoV-OC43 replication was examined in CHME3 cells transfected with 4 µg of hRTP4-2F expression vector or empty vector. The transfected CHME3 cells were infected with HCoV-OC43 at MOIs of 0.5 and 5. As a measure of virus spread, accumulation of the HCoV-OC43 N protein was monitored by immunoblot analysis of cell lysates collected over a 3-day period and synthesis of the genomic and subgenomic viral RNAs was quantified by qRT-PCR. Immunoblot analysis showed that in control cells, the N protein appeared at 24 h, increased to its peak level at 48 h and decreased at 72 h at both low and high MOI. In cells expressing hRTP4, there was no detectable N protein until 72 h at low MOI and 48 h at high MOI (Fig. 3A). Analysis of the viral RNAs showed that in the control cells, genomic and subgenomic RNAs became detectable at 24 h, continued to increase slightly at 36 and 48 h, and then decreased slightly at 72 h. The results were similar at both MOIs and for the subgenomic and genomic RNAs (Fig. 3B). In the presence of hRTP4, viral RNA synthesis was suppressed by about 100-fold for both the subgenomic and genomic RNAs at both MOIs although copy numbers increased slightly at 36 and 72 h. The findings suggest that the suppression of N protein synthesis resulted from the inhibition of viral RNA synthesis. We did not rule-out possible effects on virus entry; it is unlikely that RTP4 acts at fusion as it is localized in the endoplasmic reticulum and is not present on the plasma membrane or in endosomes.

hRTP4 is a 247 amino acid protein consisting of an N-terminal domain with three conserved 3CXXC zinc fingers (ZFD), a central domain containing a variable disordered region and a C-terminal TM anchor (Fig. 4A). The N-terminal 3CXXC ZFD has been shown to be required for antiviral activity against flaviviruses (35). To determine which of the domains are required for antiviral activity, we constructed a series of mutated FLAG-tagged hRTP4 expression vectors. In hRTP4.TMΔ22, the C-terminal TM domain was deleted by truncation of the C-terminal 22 amino acids. The three zinc fingers (ZnFs) were mutated hRTP4.ZnF1-C55S, hRTP4.ZnF2-C93S, and hRTP4.ZnF3-C154S by changing the conserved cysteine to serine. To determine the stability of the mutated and truncated proteins, 293 cells were transfected with each of the vectors or empty vector control and cell lysates were analyzed on an immunoblot probed with anti-FLAG antibody. The results showed that hRTP4.TMΔ22 and hRTP4.ZnF3-C154S were stably expressed. hRTP4.ZnF1-C55S and hRTP4.ZnF2-C93S were expressed only at low level suggesting that zinc fingers 1 and 2 are required for protein stability (Fig. 4A and B, top panel). To measure the antiviral activity of the mutated hRTP4 proteins, CHME3 cells were transfected with the wild-type (WT) and mutated hRTP4 expression vectors and the cells were then infected with HCoV-OC43. At 48 h post-infection, virus replication was measured by immunoblot analysis for the viral N protein. The results showed that the C-terminal truncated TMΔ22 and ZnF3 mutants retained antiviral activity, while ZnF1 and ZnF2 mutants were inactive (Fig. 4B, lower panel, and 4C). The results showed that the C-terminal domain and third zinc finger were dispensable consistent with the findings of Boys et al. (35). Zinc fingers 1 and 2 are required for protein stability and thus their antiviral activity could not be determined.

To further identify critical amino acid residues in the N-terminal and disordered domain, we generated a series mutated expression vectors in which 15 of the charged residues that are conserved in the two domains of human and murine RTP4 were changed to alanine (D4A, E9A, E14A, E18A, D30A, D36A, R60A, R85A, R87A, E102A, R113A E155A, K172A, K182A, and K207A) (Table S2; Fig. S2). Immunoblot analysis of the proteins in transfected 293 cells showed that all were stably expressed (Fig. 4B, lower panel, and 4C). Analysis of their antiviral activity against HCoV-OC43 was tested in transfected CHME3 cells. The results showed that the N-terminal residue mutants (D4A, E9A, E14A, E18A, D30A, D36A, R60A, R85A, R87A, and E102A) were required for antiviral activity whereas those near the C-terminus were not. Mutants toward the end in the N-terminal region and in the middle segment of hRTP4 retained antiviral function (Fig. 4B, lower panel, and 4C).

To determine whether the antiviral mechanism of hRTP4 against SARS-CoV-2 was similar to that of HCoV-OC43, we tested the antiviral activity of the mutated hRTP4s against SARS-CoV-2. For this, we transfected ACE2.CHME3 cells with the mutated expression vectors and then infected them with WA1/2020 SARS-CoV-2. At 1 and 3 dpi, genomic and subgenomic RNA levels were quantified. The results showed that the antiviral activity of the mutants against SARS-CoV-2 mirrored the activity against HCoV-OC43. The C-terminal truncated TMΔ22 and ZnF3 mutants were active, while the N-terminal ZnF1 and ZnF2 hRTP4 mutants partially lost anti-SARS-CoV-2 activity (Fig. 4D). These results suggest that hRTP4 acts by a similar mechanism against both viruses. Together, in transfected CHME3 and ACE2.CHME3 cells, antiviral activity against HCoV-OC43 and SARS-CoV-2 cells required the N-terminal domain, respectively.

The RTP family in both mouse and human consists of four members (RTP1, RTP2, RTP3, and RTP4). To determine whether the other family members have activity against CoV replication, we constructed FLAG-tagged expression vectors for the four hRTP4 family members and for mRTP4. The results showed that hRTP1, hRTP2, and hRTP3 were expressed at low to undetectable levels (Fig. 5A) and did not show any antiviral activity (not shown). mRTP4 was expressed at a level similar to that of hRTP4. Analysis of its antiviral activity against HCoV-OC43 in transfected CHME3 cells showed that the protein was inactive against the virus. Analysis of antiviral activity of mRTP4 against SARS-CoV-2 in transfected ACE2.CHME3 cells showed that mRTP4 had no effect at 1 dpi on genomic or subgenomic RNA copy number and a very slight effect, not significantly significant decrease on genomic and subgenomic RNA copy numbers at 3 dpi (Fig. 5B).

To determine the regions and amino acids of mRTP4 that are important for its function, we constructed a series of sequence modifications and point mutations of hRTP4 to substitute amino acids and groups of amino acids with those of the murine protein (Fig. 5C; Fig. S3). We generated 10 mutations in the N-terminal region, which is the domain responsible for antiviral activity (W6S, A19E, RAT22GAK, QL34VP, C37G, Q40L, RAF48TVL, WFR52RFQ, Q59C, and MPE103TPK) based on sequence alignment of the human and murine proteins (Fig. S3; Table S3). Immunoblot analysis of transfected 293 cells showed that the point mutated proteins were stably expressed (Fig. 5D, top panel). To determine the antiviral activity of the mutated proteins, CHME3 cells were transfected with each vector and then infected with HCoV-OC43. Immunoblot analysis for the viral N protein showed that the mutation in the N-terminal domain (W6S, A19E, RAT22GAK, and QL34VP) up to amino acid 34 largely maintained antiviral activity, while those between amino acids 37 and 103 which are close to ZnF1 and ZnF2 were required for antiviral function (Fig. 5D, lower panel, and 5E). The findings show that there are several amino acid positions in the murine protein that render it inactive and that the amino acid sequence of hRTP4 near the zinc fingers cannot be altered without losing antiviral activity.

Bat RTP4 was shown to interact with the flavivirus genomic RNA (35). To determine whether the human homolog binds to CoV RNA, we tested whether hRTP4 would pull-down the viral RNA. For this, we transfected CHME3 cells with FLAG-tagged hRTP4 expression vector and then infected the cells with HCoV-OC43. The cells were lysed and hRTP4 was pulled-down with anti-FLAG antibody-coated magnetic beads. Unbound complexes were removed and the bound complexes were eluted in low pH buffer. The eluted fractions were phenol extracted and the bound viral RNA was quantified by qRT-PCR. As a specificity control, the pull-down was done with cells expressing an untagged hRTP4. The results showed that the genomic and subgenomic viral RNAs bound to the FLAG-tagged hRTP4. The untagged hRTP4 was not pulled-down, confirming that pull-down was specific (Fig. 6A through C).

To determine the domains of hRTP4 required for viral RNA binding, we tested the ZFD domain mutants and the TMΔ22 carboxy-terminal truncation in the pull-down assay. The results showed that RTP4 with mutations of ZnF1 or ZnF2 bound little viral genomic and subgenomic RNAs. In contrast, the TMΔ22 and ZnF3 hRTP4 proteins retained significant viral RNA binding (Fig. 6A). The murine homolog did not bind the HCoV-OC43 RNA (Fig. 6B). To identify specific amino acids involved in viral RNA binding, we generated a series of charge-to-alanine point mutations throughout hRTP4. Analysis of these in the RNA pull-down assays showed that the mutations from amino acid 9 through 102 (E9A, E14A, E18A, D30A, D36A, R60A, R85A, R87A, and E102A) in the N-terminal region have no or drastically reduced binding with viral RNA, while those in the carboxy-terminus bind to viral RNA with high efficiency (Fig. 6C). The binding to subgenomic RNA was more pronounced than binding to genomic RNA, possibly because the assay was more efficient for smaller RNA. The results show the importance of the charge residues of the protein in the amino terminus, and suggest a role for RNA binding in antiviral activity.

K1-hACE2 [B6.129S2(Cg)ACE2tm1(ACE2)Dwnt/J] and K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J] transgenic mice were purchased from The Jackson Laboratory and bred in-house. Animal experiments were done under the protocol approved by the NYU Langone Institutional Animal Care and Use Committee (#170304) according to the standards set by the Animal Welfare Act.

SARS-CoV-2 WA1/2020 P1 virus stock (BEI Resources, NR-52281) was grown on Vero E6 cells by infection at an MOI of 0.01. At 2 h post-infection, input virus was removed and fresh medium was added. After 3 days, the virus-containing supernatant was harvested, filtered through a 0.45 µm filter, and frozen in aliquots at −80°C. Virus titers were determined by plaque assay on Vero E6 cells. The P1 stocks of Omicron BA.1 and BA.2 (BEI Resources, NR-56781) were generated by inoculating ACE2.TMPRSS2.Vero E6 cells at an MOI of 0.1. The P1 stock was expanded by a second round of replication on ACE2.TMPRSS2.Vero E6 cells infected at an MOI of 0.01. After 2 days, the supernatant was collected, filtered, and frozen at −80°C.

MRC5 cells were seeded at a density of 2 × 10⁶ /mL in 100 mm dishes. The following day, the cells were infected with 3 × 10⁶ PFU/mL of HCoV-OC43 (ATCC strain VR-1558) and incubated at 33°C for 4 days at which time 90–100% cells showed a cytopathic effect. The culture supernatant and infected cells were harvested, centrifuged at 1000 × g for 5 minutes, and the supernatant was stored at −80°C. The virus was titered by limiting dilution on MRC5 cells. Infection was scored by cytopathic effect 7 dpi in quadruplicate wells and expressed TCID₅₀/mL defined as the dose at which two out of four quadruplicate wells exhibited a cytopathic effect.

Lentiviral vector stock for ACE2 expressing lentiviral vector pLenti.ACE2 was prepared by calcium-phosphate transfection of 293T cells with pLenti.ACE2 (58) and expression plasmids pRSV-Rev, pMDL-X, and VSV-G.

Six- to eight-week-old K1-hACE2 and K18-hACE2 transgenic mice were anesthetized with by intraperitoneal injection of ketamine and xylazine and infected intranasally with 2 × 10⁴ PFU SARS-CoV-2 WA1/2020 or the same volume of PBS. At 2 dpi, the mice were killed and the lungs harvested and homogenized in Lysing Matrix D Tubes (MP Biomedicals, Irvine, California, USA) with a FastPrep-24 5G homogenizer (MP Biomedicals). The homogenates were clarified by brief centrifugation and RNA was prepared using a Quick-RNA MiniPrep kit (Zymo Research, Irvine, California, USA).

MRC5, CHME3, 293T, and Vero E6 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and incubated at 37°C under 5% CO₂. To prepare primary human monocytes, PBMCs were purified by Ficoll density gradient centrifugation from Leukopaks provided by the New York Blood Center. Monocytes were purified by plastic adherence and cultured in RPMI containing 10 mM HEPES, 24 µg/mL gentamicin, and 5% heat inactivated pooled human serum. THP-1 cells were cultured in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin and incubated at 37°C under 5% CO₂. To establish ACE2 expressing CHME3 stable cell lines, CHME3 cells were transduced with pLenti.ACE2 vector stock and after 2 days, cloned at limiting dilution in medium containing 1 µg/mL puromycin. The cell clones were stained with anti-ACE2 antibody (NOVUS) and Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (BioLegend, Eugene, Oregon, USA) and screened by flow cytometry to choose a clone that expressed high levels of cell surface ACE2. The data were analyzed with FlowJo software. To establish CHME3 ISG stable cell lines, CHME3 cells were transduced with corresponding ISG lentiviral vector stock and then selected in medium containing 1 µg/mL puromycin and screened by flow cytometry.

hRTP4 expressing lentiviral vector was generated by amplifying an hRTP4 cDNA with a forward primer containing a Spe-I restriction enzyme site and reverse primer containing 2X FLAG tag and Sal-I restriction enzyme site. The amplicon was cleaved with Spe-I and Sal-I and cloned into pLenti6.3/V5-DEST-GFP in place of GFP. hRTP4 TM domain truncation was generated using existing hRTP4 plasmid construct with a forward primer containing a Spe-I restriction enzyme site and reverse primer containing 2X FLAG tag and Sal-I restriction enzyme site. Mutations in the ZFDs were introduced by overlap extension PCR and mutations of the charged residues and the human to murine RTP4 mutants were introduced by quick change site-directed mutagenesis. Expression vectors for hRTP1, hRTP2, hRTP3, and mRTP4 were generated by amplifying the corresponding cDNA sequences (GenScript, Piscataway, New Jersey, USA) with a forward primer containing a Spe-I site and reverse primer containing 2X FLAG tag and Sal-I site and ligating to pLenti6.3/V5-DEST. The sequences of all plasmid constructs were confirmed by sequencing. The primer sequences of the constructs are shown in Table S4.

CHME3 cell lines expressing different ISGs were seeded into black 96-well plates at 90% confluency. The next day, the cells were infected with HCoV-OC43 at an MOI of 0.01 and incubated at 33°C. At 48 h post-infection, the cells were fixed with 4% PFA for 20 minutes and permeabilized by treatment for 15 minutes in 0.05% Triton X-100/PBS. The cells were blocked for 1 h in 4% FBS and then stained overnight at 4°C with anti-OC43-N antibody (Sigma-Aldrich, Damstadt, Germany). The antibody was then removed by three washes with PBS, and the cells were stained for 1 h with Alexa Fluor 594 (Invitrogen, Eugene, Oregon, USA) and DAPI at room temperature. Images were analyzed using a Cell Insight CX7 LZR high-content screening platform with HCS Navigator software for DAPI and Alexa Fluor 647.

Transfected cells were lysed in buffer containing 10 mM Tris HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail and lysate protein concentrations were determined. The cell lysates (40 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with mouse anti-FLAG antibody (Sigma-Aldrich), mouse anti-coronavirus OC43-N antibody (Sigma-Aldrich), rabbit anti-RTP4 antibody (Epigentek, Farmingdale, New York, USA), and anti-GAPDH antibody (Life Technologies, Carlsbad, California, USA) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich). The blots were washed and visualized using luminescent HRP substrate (Millipore, Burlington, MA, USA) on an iBright CL1000 imaging system.

CHME3 cells were transiently transfected with hRTP4-2F expression vector by lipofection with Lipofectamine 2000 (Invitrogen) and then infected with HCoV-OC43 at an MOI of 5. After 2 days, the transfected cells were lysed in NP-40 lysis buffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail). The cell lysates (10 µg) were incubated for 1 h at 4°C with 20 µL anti-FLAG M2 magnetic beads (Sigma-Aldrich). The beads were pulled down in a magnetic separator and washed with buffer containing 50 mM Tris HCl, pH 7.5, and 150 mM NaCl. Bound protein complexes were eluted in buffer containing 0.1 M Glycine HCl, pH 3.0. The eluted proteins were analyzed on an immunoblot probed with mouse anti-FLAG antibody (Sigma-Aldrich) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich). RNA that had been pulled down with the RTP4 was isolated by phenol extraction followed by ethanol precipitation. Genomic and subgenomic HCoV-OC43 RNAs that have been pulled down were quantified by qRT-PCR.

293T cells were seeded at 2 × 10⁵ cells/well in 6-well plate were transiently transfected with 4 µg of expression vectors along with the empty vector control using Lipofectamine 2000 (Invitrogen). After 2 days, the transfected cells were lysed in NP-40 lysis buffer and the cell lysates were analyzed on an immunoblot probed with mouse anti-FLAG antibody (Sigma-Aldrich) and anti-GAPDH antibody (Life Technologies) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich).

CHME3 cells (2 × 10⁵) were transfected with RTP4 expression vectors using Lipofectamine 2000. One dpi, the cells were infected with HCoV-OC43 at an MOI of 0.5 and 5 and incubated at 33°C. The cells were lysed on days 1 and 3 and the lysates were analyzed on an immunoblot probed with mouse anti-coronavirus OC43-N antibody (Sigma-Aldrich) followed by goat anti-mouse HRP-conjugated second antibody (Sigma-Aldrich). The infectivity for experiments was quantified by qRT-PCR. All HCoV-OC43 infections were done at Biosafety Level 2 (BSL2). SARS-CoV-2 (MOI = 0.1), Omicron BA.1, and BA.2 (MOI = 0.05, 0.5) infections in a BSL3 facility according to institutional guidelines provided by the NYU Langone and Institutional Animal Care and Use Committee according to the standards set by the Animal Welfare Act.

RNA was isolated from virus-infected CHME3 cells using Quick-RNA MiniPrep kit (Zymo Research). qRT-PCR analysis was done with TaqMan Fast Virus 1-Step Master Mix using primers in Orf1ab to amplify genomic HCoV-OC43 RNA and primers in the N gene to amplify subgenomic HCoV-OC43 RNA. SARS-CoV-2 genomic and subgenomic RNA levels were determined in SARS-CoV-2-infected ACE2.CHME3 cells and lung. Primer sequences and probe are detailed in Table S5. Relative RNA copy numbers were calculated by the comparative C_T method with GAPDH as the internal control.

RNA was prepared from 200 µL homogenized lung using the Quick-RNA MiniPrep kit (Zymo Research). cDNA was reverse-transcribed using Transcriptor RT (Roche, Mannheim, Germany) with random hexamers. qPCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, Massachusetts, USA) with a typical three-step PCR protocol. The PCR was set for 40 cycles of 95°C/15 s, 60°C/30 s, and 95°C/15 s. Signals were normalized to GAPDH, and quantification of relative gene expression was relative to untreated controls with comparative C_T method. Primer sequences are detailed in Table S5.

Statistical analyses were determined using GraphPad Prism, and statistical significance was determined with the two-tailed, unpaired t-test or two-way ANOVA. All the experiments were performed in duplicates or triplicates. Confidence intervals are shown as the mean ± SD or SEM (ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).