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Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions [1]

['Eric S. Pringle', 'Department Of Microbiology', 'Immunology', 'Dalhousie University', 'Halifax', 'Brett A. Duguay', 'Maxwell P. Bui-Marinos', 'Infectious Diseases', 'University Of Calgary', 'Calgary']

Date: 2022-11 

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro. SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.

The COVID-19 pandemic has ignited efforts to repurpose existing drugs as safe and effective antivirals. Rather than directly inhibiting viral enzymes, host-targeted antivirals inhibit host cell processes to indirectly impede viral replication and/or stimulate antiviral responses. Here, we describe a new antiviral mechanism of action for an FDA-approved thiopurine known as 6-thioguanine (6-TG). We demonstrate that 6-TG is a pro-drug that must be metabolized by host enzymes to gain antiviral activity. We show that it can inhibit the replication of human coronaviruses, including SARS-CoV-2, at least in part by interfering with the processing and accumulation of Spike glycoproteins, thereby impeding assembly of infectious progeny viruses. We provide evidence implicating host cell GTPase enzymes in the antiviral mechanism of action.

Funding: This work was supported by Canadian Institutes for Health Research (CIHR; https://cihr-irsc.gc.ca ) Project Grant PJT-148727 (to C.M.), Natural Sciences and Engineering Research Council (NSERC; https://www.nserc-crsng.gc.ca ) of Canada Discovery Grant RGPIN-2016-05083 (to S.L.B.), Coronavirus Variants Rapid Response Network (CoVaRR-Net; https://covarrnet.ca ) Grant 175622 (to J.A.C. and others), Research Nova Scotia ( https://researchns.ca ) Grant RNS-NHIG-2020-1383 and Lung Association of Nova Scotia Legacy Research Grant ( https://www.lungnspei.ca/legacy-research-grant ) (to D.K.), and Nova Scotia COVID-19 Health Research Coalition Grants ( https://researchns.ca/covid19-health-research-coalition/ ) to C.M., E.S.P., and S.L.B. SARS-CoV-2 research is supported in the laboratory of D.F. by the Canadian Institutes of Health Research (CIHR; https://cihr-irsc.gc.ca ; OV5-170349, VRI-173022 and VS1-175531). VIDO (Vaccine and Infectious Disease Organization) receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan ( https://innovationsask.ca ) and the Ministry of Agriculture ( https://www.saskatchewan.ca/government/government-structure/ministries/agriculture ) and from the Canada Foundation for Innovation ( https://www.innovation.ca ) through the Major Science Initiatives for its CL3 facility. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The emergence of SARS-CoV and MERS-CoV from animal reservoirs, followed by the current SARS-CoV-2 pandemic, has spurred efforts to discover and develop effective antivirals. These efforts have benefitted from drug repurposing strategies to identify antivirals amongst drugs that have already been approved for use in humans. We showed previously that thiopurines 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo) inhibit influenza A virus (IAV) replication by activating the UPR and interfering with the processing and accumulation of viral glycoproteins [ 34 ]. Thiopurines have been in clinical use for over 50 years to treat inflammatory diseases and cancer. They include azathioprine, 6-mercaptopurine (6-MP) and 6-TG. 6-TG is orally bioavailable and on the World Health Organization’s List of Essential Medicines. It is used to treat hematological malignancies, including acute lymphoblastic leukemia (AML), acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML), with the primary mechanism of action involving conversion into thioguanine deoxynucleotides and incorporation into cellular DNA, which preferentially kills rapidly dividing cancer cells [ 35 , 36 ]. By contrast, the anti-inflammatory effect of sub-cytotoxic doses of 6-TG for treating inflammatory bowel disease (IBD) is thought to be primarily due to inhibition of the small GTPase Rac1 [ 37 , 38 ]. This process requires 6-TG to be metabolized by the purine scavenging enzyme hypoxanthine phosphoribosyltransferase 1 (HPRT1) to yield 6-thioguanosine 5’-monophosphate (6-TGMP), which is then converted to 6-thioguanosine 5’-triphosphate (6-TGTP) by sequential phosphorylation events. 6-TGTP is the active metabolite that covalently modifies a reactive cysteine in the p-loop of Rac1. Subsequent hydrolysis of the GTPase-bound 6-TGTP adduct yields a trapped, inactive Rac1-6-TGDP product [ 37 ]. These host-targeted properties of 6-TG, combined with our knowledge of how 6-TG inhibits IAV glycoprotein processing, motivated us to investigate whether 6-TG and related thiopurines might similarly interfere with CoV glycoproteins. Moreover, we reasoned that this kind of host-targeted antiviral activity might act synergistically with the previously reported direct-acting antiviral activity of 6-TG as an inhibitor of MERS-CoV, SARS-CoV and SARS-CoV-2 papain-like cysteine proteases PL pro (nsp3) in vitro [ 39 – 42 ]. Using ectopic expression and cell culture infection models, we discovered that 6-TG and 6-TGo inhibit replication of several CoVs including SARS-CoV-2, which correlates with disruption of Spike processing, accumulation and incorporation into progeny virions. Accumulation of viral subgenomic RNAs and full-length genomes is also inhibited by these thiopurines. Finally, we were able to link the antiviral mechanism of action to the primary known mode of action of 6-TG as a pro-drug that is converted by HPRT1 into a selective inhibitor of small GTPases [ 37 , 43 , 44 ], which could be overcome using a broadly-acting GTPase agonist. Our findings extend the understanding of the mechanism of action of these candidate host-targeted antivirals and provide a means to study the impact of glycoprotein maturation on coronavirus biology, while providing the impetus for future studies of GTPases as targets for antivirals.

The striking remodeling of the ER to create replication organelles, as well as the reliance on the ER for synthesis and processing of viral glycoproteins, has prompted investigations into the effects of HCoV infection on ER stress and the unfolded protein response (UPR). Several CoVs have been shown to activate the UPR, including infectious bronchitis virus (IBV) [ 21 , 22 ], mouse hepatitis virus (MHV) [ 23 ], transmissible gastroenteritis virus (TGEV) [ 24 ], HCoV-OC43 [ 25 ] and SARS-CoV [ 26 – 28 ]. However, proximal UPR sensor activation does not always elicit downstream UPR transcription responses during CoV replication [ 29 ], which suggests complex regulation of the UPR by certain CoVs. Spike proteins from MHV [ 23 ], HCoV-HKU1 [ 27 ] and SARS-CoV [ 27 , 30 ] are sufficient for UPR activation in cell culture. Additional transmembrane CoV proteins also induce the UPR, including SARS-CoV nsp6 [ 31 ], ORF3a [ 32 ] and ORF8ab [ 33 ] proteins. Despite these discoveries, our understanding of the regulation of the UPR in CoV infection remains incomplete, and little is known about UPR regulation in SARS-CoV-2 infection.

CoV envelope proteins are processed in the secretory pathway. The viral envelope proteins Spike, Envelope (E) and Membrane (M) that are found in all HCoVs, as well as Hemagglutinin-Esterase (H-E) found in only HCoV-OC43 and HCoV-HKU1, are synthesized in the ER as transmembrane proteins [ 15 ]. CoV Spike proteins are large type I transmembrane proteins that are heavily N-glycosylated, which promotes proper folding, trimerization and trafficking [ 16 ]. Subsequent proteolytic processing is required to activate these glycosylated Spike trimers, generating the S1 attachment subunit and liberating the mature amino-terminal fusion peptide of the S2 subunit, both of which are essential components of infectious CoV virions that can enter target cells [ 17 , 18 ]. Proteolytic activation of Spike can occur in the secretory pathway of infected cells or unprocessed Spike can be incorporated into virions and Spike processing can occur during attachment and entry into naïve cells in a process that involves multiple host serine and cysteine proteases. M is the most abundant CoV envelope protein and it serves as a scaffold for viral assembly. It is a multi-spanning membrane protein, with a small lumenal amino-terminus that is N-glycosylated, three transmembrane domains, and a large cytoplasmic carboxy-terminal domain known as the endodomain that governs interactions with the other structural proteins (Spike, E and N), as well as M-M interactions [ 19 ]. E is a small type I transmembrane protein that forms pentameric complexes and functions as a viroporin, increasing the lumenal pH in the Golgi and protecting Spike from proteolysis (reviewed in [ 20 ]). The viroporin activity supports de-acidification of lysosomes and subsequent lysosomal exocytosis. M and E proteins both govern the intracellular trafficking and processing of Spike [ 19 ].

Coronaviruses (CoVs) are enveloped viruses with positive-sense, single-stranded RNA ((+)ssRNA) genomes. Seven human CoVs (HCoVs) are divided into two genera, the alphacoronaviruses (HCoV-NL63 and HCoV-229E) and betacoronaviruses (HCoV-OC43, HCoV-HKU1, MERS-CoV, SARS-CoV and SARS-CoV-2). Following entry into host cells, the (+)ssRNA CoV genome is immediately translated into polyproteins that are cleaved by viral cysteine proteases into 16 non-structural proteins (NSPs). These NSPs include the transmembrane nsp3-nsp4-nsp6 complex that generates a complex endoplasmic reticulum (ER)-derived reticulovesicular network that includes replication organelles known as double-membrane vesicles (DMVs) [ 1 – 8 ]. In DMVs, the nsp3-nsp4-nsp6 complex provides a physical anchor for the viral replicase-transcriptase complex (RTC) that replicates the large viral genome and synthesizes subgenomic mRNAs [ 9 , 10 ]. These viral RNAs traverse a crown-shaped pore to exit the DMV [ 8 ]. In the cytoplasm, viral mRNAs are translated into structural proteins; Nucleocapsid (N) is synthesized via free ribosomes in the cytoplasm, whereas mRNAs encoding envelope proteins are translated and processed in the ER and accumulate in the ER-Golgi-intermediate compartment (ERGIC). HCoV assembly takes place when full-length viral genomic RNA, coated with N, buds into the ERGIC to acquire the viral envelope and associated envelope proteins [ 11 – 13 ]. These progeny viruses traverse the Golgi apparatus and trans-Golgi network (TGN), where envelope proteins receive additional post-translational modifications, and exit the cell via lysosomal exocytosis [ 14 ].

Results

6-Thioguanine and 6-thioguanosine inhibit HCoV replication We tested the effects of three thiopurines (Fig 1A) on SARS-CoV-2 replication in Calu-3 lung adenocarcinoma cells. Treatment with 6-TG, the ribonucleoside 6-TGo, or the related thiopurine 6-MP, caused a striking 4-log decrease in the release of infectious virions after 48 h of infection, with little effect on cell viability [Selectivity Index (SI) 6-TG > 72.7, 6-TGo > 81.6, 6-MP >153.8] (Fig 1B and 1C). The sub-micromolar EC 50 values that we observed for all three thiopurines in the Calu-3 infection model were consistent with previously reported values from other groups [42,45]. 6-TG was well tolerated in three other cell lines we used for HCoV infection models (HCT-8, Huh7.5, and primary hTERT-immortalized fibroblasts) (Fig 1D). We observed that low micromolar doses of 6-TG and 6-TGo inhibited the betacoronavirus HCoV-OC43 and the alphacoronavirus HCoV-229E in their respective infection models, whereas 6-MP had no effect (Fig 1E and 1F). Because UPR stimulators thapsigargin (Tg) and tunicamycin (Tm) inhibit coronavirus replication [46–49] and our previous work demonstrated that 6-TG inhibited glycosylation of influenza A virus (IAV) glycoproteins and stimulated ER stress [34], we included Tm as a positive control in these studies. In hTERT-BJ cells, 6-TG inhibited HCoV-OC43 replication at low micromolar doses, consistent with our observations in the HCT-8 cell infection model (Fig 1G); however, HCoV-229E replication in hTERT-BJ cells was minimally affected across a range of sub-cytotoxic concentrations (Fig 1H). Together, these findings suggest that 6-TG and 6-TGo effectively inhibit replication of several coronaviruses in multiple cell types, whereas 6-MP has limited antiviral activity. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Thiopurines 6-thioguanine and 6-thioguanosine inhibit coronavirus replication. (A) Structures of thiopurines used this study in comparison to guanine. (B) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.1 then treated with 6-thioguanine (6-TG), 6-thioguanosine (6-TGo), or 6-mercaptopurine (6-MP). Supernatants were harvested after 48 h and stored at -80°C until titering on Vero’76 cells. Mock-infected cells were similarly treated with 6-TG, 6-TGo, 6-MP, or DMSO vehicle control for 48 h before testing cell viability with CellTiter 96 AQueous One (n = 3 ± SEM). Dotted line indicates Limit of Detection. (C) Summary table of 50% Cytoxic Concentration (CC 50 ), 50% Effective Concentration (EC 50 ), and Selectivity Index (SI) calculated for (A-C). (D) AlamarBlue cell viability assay of hTert-BJ, HCT-8, and Huh-7.5 cells treated with 6-TG (n = 3±SEM). (E-H) TCID50 assays for (E) HCoV-OC43 infected HCT-8 cells and (F) HCoV-229E infected Huh-7.5 cells. Cells were infected with an MOI of 0.1 then treated with tunicamycin (Tm), 6-TG, 6-TGo, 6-MP, or DMSO (n≥3 ± SEM, statistical significance was determined by one-way ANOVA). hTERT-BJ cells were infected with HCoV-OC43 (G) or HCoV-229E (H) at an MOI of 0.1 and treated with 6-TG, Tm, or DMSO. Supernatants were harvested after 23 h and stored at -80°C before titering on BHK-21 or Huh7.5 (n = 3–4 ± SEM, statistical significance was determined by one-way ANOVA). LOD = Limit of Detection for virus titer. (*, p<0.05; **, p<0.01; ns, non-significant). https://doi.org/10.1371/journal.ppat.1010832.g001

6-Thioguanine causes a coronavirus assembly defect It has been shown that co-transfection of plasmids expressing each of the four SARS-CoV-2 structural proteins allows for secretion of virus-like particles (VLPs) [19,61,62], which is concordant with earlier studies with other CoV structural proteins [63,64]. However, we observed that when Spike, E, M and N expression vectors were co-transfected into cells, 6-TG treatment not only altered the accumulation and processing of Spike, but also dramatically reduced accumulation of E and M proteins (Fig 5A). Cell supernatants were collected, filtered and pelleted in a sucrose cushion to isolate VLPs, which were then characterized by immunoblotting. We observed a dramatic reduction in VLP production from 6-TG-treated 293T cells, and these residual VLPs completely lacked Spike (Fig 5B). These findings indicate that 6-TG not only prevents Spike processing and accumulation, but also its incorporation into VLPs. We then co-transfected cells again with only three of the four Spike, E, M, and N encoding plasmids to better understand mechanisms of defective assembly. Our main conclusions are: 1) 6-TG treatment of cells expressing all structural proteins causes a strong Spike-dependent decrease in levels of E, 2) in the absence of M, the 6-TG-mediated Spike glycosylation defect is more pronounced, and 3) N is required for accumulation of E (Fig 5C). To determine if Spike from 6-TG treated cells was still functional, we employed a lentivirus pseudotyping method using Spike-Δ19, which is widely used to measure neutralizing antibody titers to SARS-CoV-2 [58,65,66]. We performed immunoblots on pseudovirions (PVs) recovered from three independent preparations that we purified by ultracentrifugation through a 20% sucrose cushion. Unlike our previous immunoblots of ectopically expressed Spike from transfected cells, we observed that most of the Spike proteins incorporated into the PVs were fully cleaved into the prominent S1 band (Fig 5D). However, 6-TG treatment led to a dramatic loss of Spike S0 and S1 species from the purified PVs, compared to the smaller decrease observed for HIV p24 levels in PV cores. We quantified PV yield from these cells by amplifying lentiviral genomes via RT-qPCR from filtered, unconcentrated cell supernatants. 6-TG treatment did not significantly reduce the quantity of capsid-protected genomes released from cells compared to vehicle control (Fig 5E). To determine the effects of 6-TG on PV infectivity, we used purified PVs to infect HEK293A cells that stably express the ACE2 receptor and measured luciferase production in the recipient cells as a measure of successful lentiviral infection. Consistent with deficient Spike incorporation into PVs, we observed that 6-TG treatment reduced PV infectivity by over 50-fold compared to PVs derived from vehicle-treated cells (Fig 5F). By contrast, PVs derived from vehicle control- and 6-TG-treated cells were equally competent to infect ACE2-deficient HEK293A cells at low levels. This suggests that 6-TG treatment not only inhibits Spike glycosylation and processing, but it also inhibits Spike trafficking and incorporation into lentiviral particles. We next investigated whether these 6-TG-mediated assembly defects altered the morphology of HCoV particles. We concentrated supernatant from HCoV-OC43 infected 293A cells by ultracentrifugation and imaged virions by transmission electron microscopy using negative staining. We observed fewer viral particles in the 6-TG treated samples, and while these particles were of similar size, we noted fewer particles with clearly discernible Spikes (Fig 5G). Taken together, the strong effects of 6-TG on Spike in a variety of models including ectopic expression, VLP production, PV production, and authentic HCoV infections, provide evidence for an additional antiviral mode of action beyond the PLpro inhibition described by others [39–42]. Indeed, we were unable to demonstrate PLpro inhibition in 293T cells in the presence of the standard 10 μM 6-TG dose used throughout this study (S2 Fig); by contrast, the known PLpro inhibitor GRL-0617 [67] prevented cleavage of a FLAG-epitope tagged NSP2/NSP3 fusion protein. PPT PowerPoint slide

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TIFF original image Download: Fig 5. 6-TG stimulates an assembly defect and inhibits Spike virion incorporation. (A) 293T cells were transfected with equal quantities of SARS-CoV-2 S, M, E, and N plasmids or empty vector (EV) then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 48 h after transfection and probed by western blotting as indicated. (B) Virus-like particles from supernatants of cells transfected in (A) were concentrated by ultracentrifugation. Samples from two independent VLP preparations were probed by western blotting as indicated. (C) 293T cells were transfected with plasmids encoding S, E, M, and N in a 1:2:2:1 ratio, substituting one of the structural proteins for EV as indicated, treated with 6-TG or DMSO then processed as in (A). (D) SARS-CoV-2 Spike pseudotyped, luciferase-expressing lentivirus particles were concentrated by ultracentrifugation. Samples from three independent lentivirus preparations were probed by western blotting as indicated. (E) Genomes from three independent lentivirus preparations were quantified by RT-qPCR (n = 3, statistical significance was determined by paired t-test; ns, non-significant). (F) 293A cells stably expressing ACE2 or empty vector control were transduced with lentivirus from three independent preparations. After 24 h, lysates were harvested and measured for luciferase activity (n = 3, statistical significance was determined by two-way ANOVA; *, p<0.05; ns, non-significant). (G) 293A cells were infected HCoV-OC43 at an MOI of 0.1, then treated with 6-TG or DMSO. Supernatants were concentrated as in (D) before virions were fixed and imaged by TEM with negative staining. Five virions from both 6-TG- and DMSO-treated samples are shown at 150,000 X magnification. Scale bar = 100 nm. Arrowhead indicates examples of Spike proteins extending from virion. https://doi.org/10.1371/journal.ppat.1010832.g005

6-Thioguanine must be processed by HPRT1 to inhibit Spike processing and coronavirus replication We observed comparable antiviral activities of 6-TG and its ribose-conjugated analogue 6-TGo throughout these studies. Metabolism of 6-TG is initiated by the purine scavenging enzyme hypoxanthine phosphoribosyltransferase 1 (HPRT1) to yield 6-thioguanosine 5’-monophosphate (6-TGMP) (Fig 6A). 6-TGMP can then be converted to 6-thioguanosine 5’-triphosphate (6-TGTP), the active form of the molecule that has been shown to form a covalent bond with a reactive cysteine in the p-loop of Rac1; subsequent hydrolysis of the covalently-bound 6-TGTP traps the GTPase in an inactive state [37]. Considering this information, we next sought to determine whether conversion of 6-TG to a ribonucleoside was required for its antiviral activity. First, we synthesized N9-methyl 6-thioguanine (6-TG-Me), which prevents conjugation to a ribose sugar due to the methyl group at N9 of the purine ring and is predicted to be resistant to conversion into 6-TGMP (Fig 6A). 6-TG-Me displayed low cytotoxicity and no antiviral activity against HCoV-OC43 across a broad range of concentrations (Fig 6B). Likewise, 6-TG-Me had no effect on SARS-CoV-2 Spike processing across a range of concentrations between 5 μM and 20 μM (Fig 6C). To corroborate this finding, we used CRISPR/Cas9 genome editing to silence expression of HPRT1 in 293T cells prior to infection with HCoV-OC43 and treatment with 6-TG or vehicle control. HPRT1 deficiency completely abrogated the antiviral activity of 6-TG and prevented defects in processing and accumulation of Spike and N (Fig 6D and 6E). HPRT1 deficiency also prevented 6-TG-mediated defects in processing and accumulation of ectopically expressed HCoV-OC43 Spike protein (Fig 6F). Together, these findings indicate that 6-TG is a pro-drug that must be metabolized by HPRT1 to convert it to an active form that can inhibit HCoV replication and glycoprotein processing. This is similar to the HPRT1 requirement for 6-TG-mediated inhibition of Rac1 GTPase [37]. Strict dependence on HPRT1 activity prompted us to investigate HPRT1 protein levels in the cell lines used in this study. We observed high HPRT1 protein levels in 293T cells and 293A cells, moderate levels in HCT-8 cells, Vero cells and Calu-3 cells, and low levels in Huh-7.5 cells and hTert-BJ cells (S3 Fig), which were unchanged by 6-TG treatment. This variability in the expression levels of HPRT1, the enzyme required to activate the 6-TG pro-drug, likely influences the antiviral efficacy of 6-TG in different cell types. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Conversion of 6-TG by HPRT1 is essential for antiviral activity and defects in Spike maturation. (A) HPRT1 catalyses a reaction between 6-TG and phosphoribosyl diphosphate (PRPP) to generate 6-thioguanosine monophosphate (6-TGMP) and pyrophosphate products. 6-TG methylated at the N9 nitrogen (6-TG-Me) was designed to be resistant to processing by HPRT1. (B) 293T cells were infected with HCoV-OC43 at an MOI of 0.1 then treated with 6-TG or 6-TG-Me. Supernatants were harvested at 24 hpi and stored at -80°C until titering on BHK-21 cells (n = 3 statistical significance was determined by paired ratio t-test; *, p<0.05; **, p<0.01; ns, non-significant). (C) 293T cells were transfected with SARS-CoV-2 Spike vector or an empty vector control then treated with 6-TG, 6-TG-Me, or DMSO vehicle control. Lysates were harvested 24 h after transfection and probed by western blotting as indicated. (D) 293T cells (parental), non-targeting (NT) CRISPR control cells, or two independent CRISPR-edited HPRT1 knockout cell lines (HPRT1-KO1 and -KO2) were infected with HCoV-OC43 at an MOI of 0.1 for 1 h prior to treatment with DMSO or 10 μM 6-TG for the remaining 23 h. Lysates were prepared 24 h and analyzed by western blotting as indicated. (E) As in (D) but cell supernatants were harvested at 24 h and titered as in (B) (n = 6 ±SEM, statistical significance was determined by paired ratio t test; ***, p<0.001; ****, p<0.0001; ns, non-significant, LOD = Limit of Detection.). (F) The cell lines in (D) were transfected with plasmids encoding codon-optimized HCoV-OC43-Spike or an empty vector followed by treatment with DMSO or 10 μM 6-TG. Lysates were prepared at 24 h and analyzed by western blotting as indicated. https://doi.org/10.1371/journal.ppat.1010832.g006

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