Abstract
INTRODUCTION: Interferons (IFNs) are cytokines that are rapidly deployed in response to invading pathogens. By initiating a signaling cascade that stimulates the expression of hundreds of genes, IFNs create an antiviral state in host cells. Because IFNs heavily influence COVID-19 outcomes, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication can be inhibited by the antiviral state, it is important to understand how the individual antiviral effectors encoded by IFN-stimulated genes (ISGs) inhibit SARS-CoV-2.
RATIONALE: We hypothesized that IFN-stimulated antiviral effectors can inhibit SARS-CoV-2, and that variation at the loci encoding these defenses underlies why some people are more susceptible to severe COVID-19.
RESULTS: We used arrayed ISG expression screening to reveal that 2′-5′-oligoadenylate synthetase 1 (OAS1) consistently inhibited SARS-CoV-2 in different contexts. Using CRISPR-Cas9, we found that endogenous OAS1 makes a substantial contribution to the antiviral state by recognizing short stretches of double-stranded RNA (dsRNA) and activating RNase L. We globally mapped where OAS1 binds to SARS-CoV-2 viral RNAs and found that OAS1 binding is remarkably specific, with two conserved stem loops in the SARS-CoV-2 5′-untranslated region (UTR) constituting the principal viral target.
OAS1 expression was readily detectable at the sites of infection in individuals who died of COVID-19, and specific OAS1 alleles are known to be associated with altered susceptibility to infection and severe disease. It had previously been reported that alleles containing a common splice-acceptor single nucleotide polymorphism in OAS1 (Rs10774671) were associated with less severe COVID-19. We determined that people with at least one allele with a G at this position could express a prenylated form of OAS1 (p46), whereas other individuals could not. Using a series of mutants, we found that C-terminal prenylation was necessary for OAS1 to initiate a block to SARS-CoV-2. Furthermore, confocal microscopy revealed that prenylation targeted OAS1 to perinuclear structures rich in viral dsRNA, whereas non-prenylated OAS1 was diffusely localized and unable to initiate a detectable block to SARS-CoV-2 replication.
The realization that prenylation is essential for OAS1-mediated sensing of SARS-CoV-2 allowed us to examine the transcriptome of infected patients and investigate whether there was a link between the expression of prenylated OAS1 and SARS-CoV-2 disease progression. Analysis of the OAS1 transcripts from 499 hospitalized COVID-19 patients revealed that expressing prenylated OAS1 was associated with protection from severe COVID-19.
Because prenylated OAS1 was so important in human cases, we wanted to determine whether horseshoe bats, the likely source of SARS-CoV-2, possessed the same defense. When we examined the genomic region where the prenylation signal should reside, retrotransposition of a long terminal repeat sequence had ablated this signal, preventing the expression of prenylated anti-CoV OAS1 in these bats.
CONCLUSION: C-terminal prenylation targets OAS1 to intracellular sites rich in viral dsRNA, which are likely the SARS-CoV-2 replicative organelles. Once in the right place, OAS1 binds to dsRNA structures in the SARS-CoV-2 5′-UTR and initiates a potent block to SARS-CoV-2 replication. Thus, the correct targeting of OAS1 and the subsequent inhibition of SARS-CoV-2 likely underpins the genetic association of alleles containing a G at Rs10774671 with reduced susceptibility to infection and severe disease in COVID-19. Moreover, the conspicuous absence of this antiviral defense in horseshoe bats potentially explains why SARS-CoV-2 is so sensitive to this defense in humans.
RATIONALE: We hypothesized that IFN-stimulated antiviral effectors can inhibit SARS-CoV-2, and that variation at the loci encoding these defenses underlies why some people are more susceptible to severe COVID-19.
RESULTS: We used arrayed ISG expression screening to reveal that 2′-5′-oligoadenylate synthetase 1 (OAS1) consistently inhibited SARS-CoV-2 in different contexts. Using CRISPR-Cas9, we found that endogenous OAS1 makes a substantial contribution to the antiviral state by recognizing short stretches of double-stranded RNA (dsRNA) and activating RNase L. We globally mapped where OAS1 binds to SARS-CoV-2 viral RNAs and found that OAS1 binding is remarkably specific, with two conserved stem loops in the SARS-CoV-2 5′-untranslated region (UTR) constituting the principal viral target.
OAS1 expression was readily detectable at the sites of infection in individuals who died of COVID-19, and specific OAS1 alleles are known to be associated with altered susceptibility to infection and severe disease. It had previously been reported that alleles containing a common splice-acceptor single nucleotide polymorphism in OAS1 (Rs10774671) were associated with less severe COVID-19. We determined that people with at least one allele with a G at this position could express a prenylated form of OAS1 (p46), whereas other individuals could not. Using a series of mutants, we found that C-terminal prenylation was necessary for OAS1 to initiate a block to SARS-CoV-2. Furthermore, confocal microscopy revealed that prenylation targeted OAS1 to perinuclear structures rich in viral dsRNA, whereas non-prenylated OAS1 was diffusely localized and unable to initiate a detectable block to SARS-CoV-2 replication.
The realization that prenylation is essential for OAS1-mediated sensing of SARS-CoV-2 allowed us to examine the transcriptome of infected patients and investigate whether there was a link between the expression of prenylated OAS1 and SARS-CoV-2 disease progression. Analysis of the OAS1 transcripts from 499 hospitalized COVID-19 patients revealed that expressing prenylated OAS1 was associated with protection from severe COVID-19.
Because prenylated OAS1 was so important in human cases, we wanted to determine whether horseshoe bats, the likely source of SARS-CoV-2, possessed the same defense. When we examined the genomic region where the prenylation signal should reside, retrotransposition of a long terminal repeat sequence had ablated this signal, preventing the expression of prenylated anti-CoV OAS1 in these bats.
CONCLUSION: C-terminal prenylation targets OAS1 to intracellular sites rich in viral dsRNA, which are likely the SARS-CoV-2 replicative organelles. Once in the right place, OAS1 binds to dsRNA structures in the SARS-CoV-2 5′-UTR and initiates a potent block to SARS-CoV-2 replication. Thus, the correct targeting of OAS1 and the subsequent inhibition of SARS-CoV-2 likely underpins the genetic association of alleles containing a G at Rs10774671 with reduced susceptibility to infection and severe disease in COVID-19. Moreover, the conspicuous absence of this antiviral defense in horseshoe bats potentially explains why SARS-CoV-2 is so sensitive to this defense in humans.
| Original language | English |
|---|---|
| Article number | eabj3624 |
| Number of pages | 18 |
| Journal | Science |
| Volume | 374 |
| Issue number | 6567 |
| DOIs | |
| Publication status | Published - 28 Oct 2021 |
Bibliographical note
Funding:This work was partly funded by UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC MR/V028448/1 to M.P. and S.J.W.) and the MRC through the following grants: MR/ K024752/1(to S.J.W.), MC_UU_12014/10 (to M.P. and S.J.W.), MC_UU_12014/12 (to J.H., D.L.R., and S.K.) and MR/P022642/1 (to S.J.W. and S.J.R.), MR/V000489/1 (to E.C.Y.W. and R.J.S.), MR/S00971X/1 (to R.J.S. and E.C.Y.W.), MR/P001602/1 (to E.C.Y.W.), and MR/V011561/1 (to P.J.L.). Support was also provided by a Wellcome Principal Research Fellowship 210688/Z/18/Z (to P.J.L.), a Wellcome Trust Fellowship 201366/Z/16/Z (to S.J.R.), a Wellcome Investigator Award 209412/Z/17/Z (to I.D.), the Addenbrooke’s Charitable Trust and the NIHR Cambridge Biomedical Research Centre (to P.J.L.), support from the German Research Foundation, Deutsche Forschungsgemeinschaft; project number 406109949 (to V.H.), and German Federal Ministry of Food and Agriculture through BMEL Förderkennzeichen: 01KI1723G (to V.H.), and a Daphne Jackson Fellowship funded by Medical Research Scotland (to S.S.). A.C. is supported by MRC grants MR/R021562/1, MC_UU_12014/10, and MC_UU_12014/12. J.Y.L. is funded by a Medial Sciences Graduate Studentship, University of Oxford. L.I. is funded by BBSRC DTP scholarship number BB/M011224/1. MAIC analysis was supported by the SHIELD Consortium (MRC grant MRNO2995X/1). ISARIC4C is supported by grants from the Medical Research Council (grant MC_PC_19059), the NIHR (award CO-CIN-01) and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (award 200907), NIHR HPRU in Respiratory Infections at Imperial College London with PHE (award 200927), Wellcome Trust and Department for International Development (215091/Z/18/Z), and the Bill and Melinda Gates Foundation (OPP1209135), and Liverpool Experimental Cancer Medicine Centre (C18616/A25153), NIHR Biomedical Research Centre at Imperial College London (IS-BRC-1215-20013), EU Platform foR European Preparedness Against (Re-)emerging Epidemics (PREPARE) (FP7 project 602525) and NIHR Clinical Research Network provided infrastructure support for this research.