Inhibition of SARS–CoV-2 by type I and type III interferons

Abstract

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS–CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS–CoV-2 and compared them with those against SARS–CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS–CoV-2. In contrast, SARS–CoV-1 was restricted only by IFN-α in these cell lines. SARS–CoV-2 generally exhibited a broader IFN sensitivity than SARS–CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS–CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS–CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect–prone type III IFN are good candidates for the management of COVID-19. The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS–CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS–CoV-2 and compared them with those against SARS–CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS–CoV-2. In contrast, SARS–CoV-1 was restricted only by IFN-α in these cell lines. SARS–CoV-2 generally exhibited a broader IFN sensitivity than SARS–CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS–CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS–CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect–prone type III IFN are good candidates for the management of COVID-19. The massive pandemic caused by coronavirus SARS–CoV-2 (1Coronaviridae Study Group of the International Committee on Taxonomy of VirusesThe species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS–CoV-2.Nat. Microbiol. 2020; 5 (32123347): 536-54410.1038/s41564-020-0695-zCrossref PubMed Scopus (4266) Google Scholar, 2Wu A. Peng Y. Huang B. Ding X. Wang X. Niu P. Meng J. Zhu Z. Zhang Z. Wang J. Sheng J. Quan L. Xia Z. Tan W. 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Guarding the frontiers: the biology of type III interferons.Nat. Immunol. 2015; 16 (26194286): 802-80910.1038/ni.3212Crossref PubMed Scopus (222) Google Scholar, 5Ye L. Schnepf D. Staeheli P. Interferon-λ orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19 (31201377): 614-62510.1038/s41577-019-0182-zCrossref PubMed Scopus (128) Google Scholar). Both type I and type III IFNs are clinically used or being tested, respectively, against a range of ailments that include viral diseases (6O'Brien T.R. Young H.A. Donnelly R.P. Prokunina-Olsson L. Interferon lambda: disease impact and therapeutic potential.J. Interferon Cytokine Res. 2019; 39 (30998425): 586-59110.1089/jir.2019.0018Crossref PubMed Scopus (6) Google Scholar, 7Snell L.M. McGaha T.L. Brooks D.G. Type I interferon in chronic virus infection and cancer.Trends Immunol. 2017; 38 (28579323): 542-55710.1016/j.it.2017.05.005Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Previously, we and others have demonstrated the potential of IFNs to inhibit the two related, previously emerged pathogenic coronaviruses SARS–CoV-1 and MERS-CoV (8Chan R.W.Y. Chan M.C.W. Agnihothram S. Chan L.L.Y. Kuok D.I.T. Fong J.H.M. Guan Y. Poon L.L.M. Baric R.S. Nicholls J.M. Peiris J.S.M. Tropism of and innate immune responses to the novel human betacoronavirus lineage C virus in human ex vivo respiratory organ cultures.J. Virol. 2013; 87 (23552422): 6604-661410.1128/JVI.00009-13Crossref PubMed Scopus (145) Google Scholar, 9Cinatl J. Morgenstern B. Bauer G. Chandra P. Rabenau H. Doerr H.W. Treatment of SARS with human interferons.Lancet. 2003; 362 (12892961): 293-29410.1016/S0140-6736(03)13973-6Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 10Falzarano D. de Wit E. Martellaro C. Callison J. Munster V.J. Feldmann H. Inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin.Sci. 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Strong J.E. Aoki F. Plummer F. Jones S.M. Feldmann H. Severe acute respiratory syndrome related coronavirus is inhibited by interferon-alpha.J. Infect. Dis. 2004; 189 (15031783): 1164-116710.1086/382597Crossref PubMed Scopus (179) Google Scholar, 15Zielecki F. Weber M. Eickmann M. Spiegelberg L. Zaki A.M. Matrosovich M. Becker S. Weber F. Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus.J. Virol. 2013; 87 (23449793): 5300-530410.1128/JVI.03496-12Crossref PubMed Scopus (121) Google Scholar). Here, we investigated the potential of type I and type III IFNs against the newly emerged SARS–CoV-2. We tested the effect of type I IFN against SARS–CoV-2 compared with the SARS–CoV-1 from 2003. Two different cell lines were employed, namely the human bronchial epithelial Calu-3 and the primate kidney epithelial Vero E6. The cells were first treated for 16 h with 100, 500, or 1000 units/ml of recombinant human IFN-α(B/D) and then infected with the viruses at a multiplicity of infection (MOI) of 0.01 plaque forming units (PFU)/cell to obtain multistep growth. Virus titers in supernatants were determined 24 h later, when titers are reaching a plateau (see below). The data of three biological replicates are shown in Fig. 1. Because several titers were below the detection limit of our plaque assay, a rank correlation test (Spearman's exact rank correlation test) was used for statistical dose-response correlation analysis. For SARS–CoV-2 (dark gray bars), statistically significant negative correlation coefficients (CC) were obtained for both cell lines, indicating that viral replication is increasingly inhibited by IFN-α. For SARS–CoV-1 (light gray bars), titers were also affected. However, at least in Vero E6 cells, the reduction of SARS–CoV-1 appears to be weaker than the reduction of SARS–CoV-2 (Fig. 1B). Observations were similar when the input MOI was reduced to 0.001 (Fig. S1), except that titers of SARS–CoV-1 in Calu-3 cells were already very low in the absence of any IFN-α, resulting in a nonsignificant effect of additional IFN. These data may suggest that the potency of IFN to reduce viral titers may be stronger and more consistent against SARS–CoV-2 than against SARS–CoV-1. To further investigate the potential differences between the viruses, we repeated the experiment three times more with the intermediate dose of 100 units/ml and analyzed the data statistically after pooling them with the previous three replicates. Two-way ANOVA was used to simultaneously evaluate the influence of both IFN-α and virus species on virus reduction. This analysis (Fig. 2, A and B) showed again that (i) both viruses are reduced by IFN (comparison of 0 versus 100 units/ml IFN-α, p(IFN)) and (ii) there are differences between the SARS–CoV species (comparison of the virus experiments, p(virus)). Moreover, the “interaction” p value showed that, at least in Vero cells, the degree of IFN sensitivity depends on the virus species, again indicating that SARS–CoV-2 is more IFN-sensitive than SARS–CoV-1.Figure 2Sensitivity of SARS–CoV-2 and SARS–CoV-1 to intermediate-dose type I IFN. Calu-3 (A) and Vero E6 cells (B) were pretreated with 100 units/ml IFN-α, infected at an MOI of 0.01, and titrated 24 h later. Log-transformed data were analyzed by two-way ANOVA with factors “IFN” and “virus,” for each of which the specific p values are indicated. p (interaction) designates the probability that IFN sensitivity depends on the virus species. Data points and geometric mean values from six independent experiments are shown. Note that three of the six biological repeats are repeats from Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The primary tropism of coronaviruses typically involves epithelia of the respiratory and gastrointestinal tracts (16Hulswit R.J.G. de Haan C.A.M. Bosch B.J. Coronavirus spike protein and tropism changes.Adv. Virus Res. 2016; 96 (27712627): 29-5710.1016/bs.aivir.2016.08.004Crossref PubMed Scopus (263) Google Scholar). On such mucosal barriers, type III IFNs rather than type I IFNs are the predominant antiviral cytokine (4Wack A. Terczyńska-Dyla E. Hartmann R. Guarding the frontiers: the biology of type III interferons.Nat. Immunol. 2015; 16 (26194286): 802-80910.1038/ni.3212Crossref PubMed Scopus (222) Google Scholar, 5Ye L. Schnepf D. Staeheli P. Interferon-λ orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19 (31201377): 614-62510.1038/s41577-019-0182-zCrossref PubMed Scopus (128) Google Scholar). Although the IFN induction as well as signaling and up-regulation of IFN-stimulated genes (ISGs) are very similar, type III IFNs engage a different receptor that is restricted to epithelial cells, and generate a weaker but longer-lasting antiviral response (5Ye L. Schnepf D. Staeheli P. Interferon-λ orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19 (31201377): 614-62510.1038/s41577-019-0182-zCrossref PubMed Scopus (128) Google Scholar, 17Pervolaraki K. Rastgou Talemi S.R. Albrecht D. Bormann F. Bamford C. Mendoza J.L. Garcia K.C. McLauchlan J. Höfer T. Stanifer M.L. Boulant S. Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance.PLoS Pathog. 2018; 14 (30485383): e100742010.1371/journal.ppat.1007420Crossref PubMed Scopus (64) Google Scholar). IFN-λ was previously shown to have activity against coronaviruses (11Kindler E. Jonsdottir H.R. Muth D. Hamming O.J. Hartmann R. Rodriguez R. Geffers R. Fouchier R.A. Drosten C. Muller M.A. Dijkman R. Thiel V. Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential.mBio. 2013; 4 (23422412): e00611-e0061210.1128/mBio.00611-12Crossref PubMed Scopus (158) Google Scholar, 18Hamming O.J. Terczyńska-Dyla E. Vieyres G. Dijkman R. Jørgensen S.E. Akhtar H. Siupka P. Pietschmann T. Thiel V. Hartmann R. Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses.EMBO J. 2013; 32 (24169568): 3055-306510.1038/emboj.2013.232Crossref PubMed Scopus (156) Google Scholar, 19Mordstein M. Neugebauer E. Ditt V. Jessen B. Rieger T. Falcone V. Sorgeloos F. Ehl S. Mayer D. Kochs G. Schwemmle M. Günther S. Drosten C. Michiels T. Staeheli P. Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections.J. Virol. 2010; 84 (20335250): 5670-567710.1128/JVI.00272-10Crossref PubMed Scopus (319) Google Scholar) and proposed as potential COVID-19 treatment (20Prokunina-Olsson L. Alphonse N. Dickenson R.E. Durbin J.E. Glenn J.S. Hartmann R. Kotenko S.V. Lazear H.M. O'Brien T.R. Odendall C. Onabajo O.O. Piontkivska H. Santer D.M. Reich N.C. Wack A. et al.COVID-19 and emerging viral infections: The case for interferon lambda.J. Exp. Med. 2020; 217 (32289152): e2020065310.1084/jem.20200653Crossref PubMed Scopus (134) Google Scholar). Hence, we compared the sensitivity of the two SARS–coronaviruses also to recombinant human IFN-λ. As shown in Fig. 3A, pretreatment with 10 or 100 ng/ml IFN-λ exhibited only in Vero E6 cells a dose-dependent inhibitory effect on SARS–CoV-2. For SARS–CoV-1, by contrast, no significant inhibition was noted in any of the cell lines. To further investigate the difference between the viruses, we repeated the IFN-λ experiment three times more with the intermediate dose of 10 ng/ml and analyzed the data after pooling with the previous 10 ng/ml IFN-λ experiment (Fig. 3B). Conventional statistical analysis (one-tailed Student's t test, because none of the values was below the detection limit) again revealed a significant impact of IFN-λ on SARS–CoV-2 and the lack of an effect for SARS–CoV-1. Our data thus show that IFN-λ can inhibit SARS–CoV-2 but not SARS–CoV-1. A recent study on the host cell interactome of SARS–CoV-2 a of human for which and drugs are M. J. K. R. B. et SARS–CoV-2 protein for 2020; Scopus Google Scholar). a to target the type I and type III IFN-triggered JAK/STAT signaling pathway S. P. L.M. G. M. analysis of inhibitor PubMed Scopus Google was the proposed of cell interactions M. J. K. R. B. et SARS–CoV-2 protein for 2020; Scopus Google Scholar). Because virus inhibition by an IFN inhibitor we to the influence of on SARS–CoV-2 were pretreated with for 16 h and infected at the two different and titers were 24 or h later. As shown in Fig. with titers in are already reaching a plateau at the In Calu-3 cells, a effect on SARS–CoV-2 at h and at both MOI 0.01 and 0.001 (Fig. and Fig. contrast, in Vero E6 there was a a negative effect (Fig. and Fig. Calu-3 cells are of IFN in response to virus Vero cells are not F. Weber M. Eickmann M. Spiegelberg L. Zaki A.M. Matrosovich M. Becker S. Weber F. Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus.J. Virol. 2013; 87 (23449793): 5300-530410.1128/JVI.03496-12Crossref PubMed Scopus (121) Google Scholar). Our data thus that (i) is an rather than an inhibitor of SARS–CoV-2 and (ii) the effect is to inhibition of the antiviral JAK/STAT signaling because it is not in the IFN Vero E6 cells. Our suggest that SARS–CoV-2 is more sensitive to IFNs than SARS–CoV-1. Moreover, type I IFN to have a more effect than type III IFN. To differences in signaling or for these we tested the of the cell lines to to the IFNs. The analysis (Fig. that Calu-3 cells have a very similar to both of IFN of and and of the IFN-stimulated MxA and Vero E6 cells also to IFN-λ as M. J. interferon induction of in response to virus Virol. 2010; 84 PubMed Scopus Google but the response was than to IFN-α. Moreover, in Calu-3 cells, there was already a which was not in Vero cells. was in able to influence these as but it was more against IFN-λ than against IFN-α, and its on IFN-stimulated genes were more in the Vero E6 compared with the Calu-3 (Fig. both cell lines are to to the different of but IFN-λ was less which is in with our on SARS–CoV as well as with previous (5Ye L. Schnepf D. Staeheli P. Interferon-λ orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19 (31201377): 614-62510.1038/s41577-019-0182-zCrossref PubMed Scopus (128) Google Scholar, 17Pervolaraki K. Rastgou Talemi S.R. Albrecht D. Bormann F. Bamford C. Mendoza J.L. Garcia K.C. McLauchlan J. Höfer T. Stanifer M.L. Boulant S. Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance.PLoS Pathog. 2018; 14 (30485383): e100742010.1371/journal.ppat.1007420Crossref PubMed Scopus (64) Google Scholar). The recently emerged SARS–CoV-2 is for the Here, we show that type I and type III IFNs are able to inhibit SARS–CoV-2 with that in our were more than against the SARS–CoV-1 from 2003. be noted that the differences between the viruses be to the cell used or to the differences in virus replication in of IFN the SARS–CoV-2 is more sensitive to IFNs to be IFN-α was the of against chronic infection with C virus the recent of antiviral drugs M.L. C. G. D. M. G. D. A. A. J. J. for chronic C virus J. Med. PubMed Scopus Google Scholar). Although with IFN-α is used to of is and is IFN-λ I and clinical with C virus S. G. R. J. R. T. S. T. J. M.A. L. et study of for the treatment of chronic HCV Full Text Full Text PDF PubMed Scopus Google Scholar). exhibited as well as but the III were because of the of IFN-λ as because of its to mucosal and the less but more antiviral response it (5Ye L. Schnepf D. Staeheli P. Interferon-λ orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19 (31201377): 614-62510.1038/s41577-019-0182-zCrossref PubMed Scopus (128) Google Scholar, 17Pervolaraki K. Rastgou Talemi S.R. Albrecht D. Bormann F. Bamford C. Mendoza J.L. Garcia K.C. McLauchlan J. Höfer T. Stanifer M.L. Boulant S. Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance.PLoS Pathog. 2018; 14 (30485383): e100742010.1371/journal.ppat.1007420Crossref PubMed Scopus (64) Google Scholar). In with our a of show that also others found type I and type III IFNs to be against SARS–CoV-2 replication in cell A. C. R. SARS–CoV-2 is sensitive to type I interferon 2020; Scholar, N. J. S. Huang C. antiviral activities of type I interferons to SARS–CoV-2 2020; Scholar, M.L. C. M. S. M. T. R. 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B. et SARS–CoV-2 protein for 2020; Scopus Google Scholar, J. A. C. O. D. P. antiviral and Infect. Dis. 2020; Full Text Full Text PDF PubMed Scopus Google and a clinical is case were V. C. S. V. A. of in with SARS–CoV-2 two case J. 2020; PubMed Scopus Google Scholar). The replication obtained with on the IFN-competent Calu-3 cells that is not at SARS–CoV-2 drugs that with viral host may not be antiviral but rather the Calu-3 and Vero E6 cells were in with in a at SARS–CoV-2 SARS–CoV-2 C. M. P. G. G. C. T. Thiel V. C. W. M. Drosten C. P. K. S. et of 2019-nCoV infection from an in J. Med. 2020; PubMed Scopus Google Scholar) and SARS–CoV-1 C. S. W. S. H.R. Becker S. Rabenau H. M. L. Fouchier R.A. A. A.M. J. Eickmann M. N. et of a novel coronavirus in with severe acute respiratory J. Med. 2003; PubMed Scopus Google Scholar) were on Vero E6 cells and via The viruses were titrated on Vero E6 cells. experiments were 3 with respiratory cells were tested The cells were pretreated for 16 h with the of IFN-α(B/D) M.A. de K. A recombinant human with a Virol. PubMed Scopus (72) Google recombinant O.J. Terczyńska-Dyla E. Vieyres G. Dijkman R. Jørgensen S.E. Akhtar H. Siupka P. Pietschmann T. Thiel V. Hartmann R. Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses.EMBO J. 2013; 32 (24169568): 3055-306510.1038/emboj.2013.232Crossref PubMed Scopus (156) Google Scholar, M. Hartmann R. The influence of the on activity and Interferon Cytokine Res. 2019; 39 PubMed Scopus Google or with were at a MOI of 0.01 and the times cell supernatants were and titrated by plaque on Vero E6 cells. The cells were treated for 24 h with the of IFNs or h and in protein inhibitor and were on and to via in with detection of and or primary was at 4 The were in with with for and again in with and in the were with a and the were a system The primary used were and The used were and The statistical analysis of the data were by of the statistical of Scholar) and For the statistical of the dose-response effect of IFN I and against the were not because of several values below the detection limit and in the of the rank was used in the exact Because the was reduction of p values were only two IFN were to with no data below the detection then the t test for independent was used For the effect of IFN and virus type the two-way ANOVA was especially a between the two tested In the statistical as well as the the response was because of its statistical In a statistical of was data and are the Download with analysis of correlation coronavirus coronavirus disease interferon Janus respiratory syndrome plaque forming units severe acute respiratory syndrome transducer and activator of transcription multiplicity of infection IFN-stimulated

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