In a recent study published in PLoS Pathogens, researchers described a novel antiviral mechanism of action for an FDA (food and drug administration)-approved thiopurine known as 6-thioguanine (6-TG).
Background
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has spurred efforts to repurpose drugs to develop effective and safe antivirals. Host-targeted antivirals (HTAs) indirectly inhibit viral replication by inhibiting host cellular processes and/or stimulating antiviral responses.
The authors of the present study previously demonstrated that thiopurines, 6-thioguanosine (6-TGo) and 6-TG inhibit IAV (influenza A virus) replication by unfolded protein response (UPR) activation and interfering with the viral glycoprotein processing and accumulation.
About the study
The present study investigated whether 6-TG and other thiopurines could interfere with coronavirus (CoV) glycoproteins.
Thiopurine efficacy against severe SARS-CoV-2, human CoV (HCoV)-OC43 and HCoV-229E replication inhibition and disruption of viral ribonucleic acid (RNA) synthesis were assessed. Cell culture experiments were performed using 293T cells, HCT-8 (human ileocecal adenocarcinoma cell line), Huh7.5 (human hepatoma-derived cell line), primary human telomerase reverse transcriptase (hTERT)-immortalized fibroblast and Calu-3 (lung adenocarcinoma cell line) cells.
Further, viral particle release was assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of extracellular viral genomes. Immunostaining analysis using HCoV-OC43-infected cells was performed for anti-nucleocapsid (N) antibodies and double-stranded RNA (dsRNA). Ribopuromycinylation assays were performed, and 293T cells were transfected with plasmids encoding SARS-CoV-2 structural proteins such as N, spike (S), membrane (M), and envelope (E) proteins to evaluate the impact of 6-TG on SARS-CoV-2 structural proteins.
Further, SARS-CoV-2 S was ectopically expressed and assessed against multiple 6-TG concentrations, following which immunoblotting analysis with pseudovirions (PV) was performed. S-expressing 293T cell lysates were treated with PNGase F (peptide-N-glycosidase) to eliminate N-linked glycans from polypeptide chains, and the effects of 6-TG on the secretory pathway were assessed by Gaussia luciferase assays.
Flow cytometry (FC) analysis and surface staining of S-expressing 293T cells were performed to measure S secretion, and the cells were co-transfected with EGFP+ (enhanced green fluorescent protein) plasmids to assess alterations in S protein accumulation.
Furthermore, cell co-transfections with SARS-CoV-2 structural proteins and plasmids were performed to elucidate the defective assembly mechanisms, and the team determined if any known 6-TG targets cause S protein maturation defects. The team also assessed potential HCoV morphological alterations after 6-TG treatment by transmission electron microscopy (TEM) analysis.
Results
6-TG inhibited the initial stage of SARS-CoV-2 and HCoV-OC43 replication, limiting full-length viral genome, structural proteins and subgenomic RNA accumulation. Ectopic S expression analysis showed enhanced S protein electrophoretic mobility from several βCoVs by 6-TG treatment, in accordance with the in vitro enzymatic N-linked oligosaccharide elimination from the S protein. SARS-CoV-2 VLPs (virus-like particles) in 6-TG-treated cells lacked the S protein.
Similar 6-TG effects were observed on SARS-CoV-2 S-pseudotyped lentivirus production yielding S-deficient pseudoviruses that could not infect angiotensin-converting enzyme 2 (ACE2)-expressing cells. The findings indicated that 6-TG treatment led to defective S protein processing and trafficking and thereby impeding infectious progeny virus assembly. However, the conversion of 6-TG to its nucleotide (nt) form by HPRT1 (hypoxanthine phosphoribosyltransferase 1) was essential for antiviral activity, which could be overcome by ML099, a guanosine-5′-triphosphate (GTP)ase agonist.
No GTPase inhibitors [Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog family member A (RhoA), and Cell division control protein 42 homolog (CDC42)] affected S accumulation or processing, indicating that 6-TG inhibited S maturation by inhibiting an unknown cellular GTPase. 6-TG, 6-thioguanosine (6-TGo), and 6-mercaptopurine (6-MP) caused four-log reductions in SARS-CoV-2 virion release and 6-TGo showed comparable HCoV-OC43 and HCoV-229E inhibition, whereas 6-MP was ineffective. The RT-qPCR analysis showed that 6-TG treatment reduced HCOV-OC43 titers by 20-fold on the initial day of infection.
Putative full-length genomic viral RNA was reduced by 10-fold in most stages of infection. 6-TG treatment caused similar reductions in viral S- and N-encoding sub-genomic RNA correlating with lower protein accumulation. Immunostaining HCoV-OC43-infected cells with anti-N antibodies showed punctate staining initially and peripheral staining subsequently. Post-6-TG treatment, the stained areas were brighter, with large puncta observed after 24 hours post-infection (hpi). Immunostaining for dsRNA showed considerably reduced dsRNA signals among 6-TG-treated cells.
6-TG delayed or suppressed UPR downstream transcriptional responses but did not affect the translation initiation rates in HCoV-OC43-infected cells. 6-TG inhibition of HCoV-OC43 infection restricted inositol-requiring enzyme 1 (IRE1) activation and X-box binding protein 1 (XBP1s) target gene accumulation. The findings indicated that even though 6-TG interfered with viral full-length genomic and subgenomic RNA synthesis, host shutoff was unperturbed.
6-TG reduced SARS-CoV-2 structural protein (especially S) expression and immunoblotting analysis showed a high molecular weight S-band denotive of the S0 precursor protein, which was also sensitive to PNGaseF treatment. The electrophoretic mobility analysis findings indicated that 6-TG inhibited S glycosylation and processing, and Gaussia luciferase experiments showed that 6-TG didn’t cause global secretory pathway disruption. The TEM analysis showed fewer viral particles in 6-TG-treated cells.
Conclusion
Overall, the study findings highlighted small GTPases as potential HTA targets, and the effects of 6-TG on S in several models, such as ectopic expression, authentic HCoV infections, and production of PVs and VLPs, indicate an antiviral mechanism beyond papain-like protease (PLpro) inhibition.
