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Early Temporal Dynamics of Cellular Responses to SARS-Cov-2

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Early Temporal Dynamics of Cellular Responses to SARS-Cov-2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.158154; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Early temporal dynamics of cellular responses to SARS-CoV-2 2 Arinjay Banerjee1,2, Patrick Budylowski3, Daniel Richard4, Hassaan Maan5,6, Jennifer A. 3 Aguiar7, Nader El-Sayes2, Michael R. D’Agostino2, Benjamin J.-M. Tremblay7, Sam Afkhami2, 4 Mehran Karimzadeh5,6,8, Lily Yip9, Mario Ostrowski10, Jeremy A. Hirota2,11, Robert Kozak9,12, 5 Terence D. Capellini4, Matthew S. Miller2,13, Andrew G. McArthur2,13, Bo Wang5,6,8, Andrew C. 6 Doxey7,11, Samira Mubareka9,12 and Karen Mossman1,2,* 7 8 1Department of Pathology and Molecular Medicine, McMaster University; Hamilton, ON, 9 Canada. 10 2Michael G. DeGroote Institute for Infectious Disease Research, McMaster University; 11 Hamilton, ON, Canada. 12 3Institute of Medical Science, University of Toronto, Toronto, ON, Canada 13 4Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, USA. 14 5Vector Institute for Artificial Intelligence, Toronto, ON, Canada 15 6Peter Munk Cardiac Centre, University Health Network, Toronto, ON, Canada. 16 7Department of Biology, University of Waterloo; Waterloo, Ontario, N2L 3G1; Canada 17 8Department of Medical Biophysics, University of Toronto, ON, Canada 18 9Sunnybrook Research Institute, Toronto, ON, Canada 19 10Department of Medicine, University of Toronto, Toronto, ON, Canada 20 11Division of Respirology, Department of Medicine, McMaster University, Hamilton, ON, 21 Canada. 22 12Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, 23 Canada. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.158154; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 13Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, 25 Canada. 26 27 28 *Corresponding author: 29 Dr. Karen Mossman 30 Email: [email protected] 31 Classification: Biological Sciences; Microbiology 32 Keywords: SARS-CoV-2, transcription, interferon, ISGs, coronavirus 33 Author contributions: A.B., D.R., H.M., A.G.M. and J.A.A. designed the study; A.B., P.B., 34 N.E-.S., M.R.D., S.A. and L.Y. performed research; A.B., D.R., H.M., J.A.A, N.E-.S., B.J.-M.T., 35 and M.K. analyzed the data; J.A.H. and M.S.M. provided reagents; A.B., M.O., J.A.H., R.K., 36 T.C., M.S.M., A.G.M, A.C.D., S.M. and K.M. provided funding and supervised the study; A.B., 37 D.R., H.M., N.E-.S., J.A.A. and A.G.M. wrote the manuscript. All authors reviewed the 38 manuscript. 39 40 41 42 43 44 45 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.158154; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 46 Abstract 47 Two highly pathogenic human coronaviruses that cause severe acute respiratory syndrome 48 (SARS) and Middle East respiratory syndrome (MERS) have evolved proteins that can inhibit 49 host antiviral responses, likely contributing to disease progression and high case-fatality rates. 50 SARS-CoV-2 emerged in December 2019 resulting in a global pandemic. Recent studies have 51 shown that SARS-CoV-2 is unable to induce a robust type I interferon (IFN) response in human 52 cells, leading to speculation about the ability of SARS-CoV-2 to inhibit innate antiviral 53 responses. However, innate antiviral responses are dynamic in nature and gene expression levels 54 rapidly change within minutes to hours. In this study, we have performed a time series RNA-seq 55 and selective immunoblot analysis of SARS-CoV-2 infected lung (Calu-3) cells to characterize 56 early virus-host processes. SARS-CoV-2 infection upregulated transcripts for type I IFNs and 57 interferon stimulated genes (ISGs) after 12 hours. Furthermore, we analyzed the ability of 58 SARS-CoV-2 to inhibit type I IFN production and downstream antiviral signaling in human 59 cells. Using exogenous stimuli, we discovered that SARS-CoV-2 is unable to modulate IFNβ 60 production and downstream expression of ISGs, such as IRF7 and IFIT1. Thus, data from our 61 study indicate that SARS-CoV-2 may have evolved additional mechanisms, such as masking of 62 viral nucleic acid sensing by host cells to mount a dampened innate antiviral response. Further 63 studies are required to fully identify the range of immune-modulatory strategies of SARS-CoV- 64 2. 65 Significance 66 Highly pathogenic coronaviruses that cause SARS and MERS have evolved proteins to 67 shutdown antiviral responses. The emergence and rapid spread of SARS-CoV-2, along with its 68 relatively low case-fatality rate have led to speculation about its ability to modulate antiviral 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.158154; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 69 responses. We show that SARS-CoV-2 is unable to block antiviral responses that are mounted by 70 exogenous stimuli. Data from our study provide promising support for the use of recombinant 71 type I IFN as combination therapy to treat COVID-19 patients. Furthermore, our data also 72 suggest that the inability of SARS-CoV-2 to efficiently modulate antiviral responses may be 73 associated with its low case-fatality rate compared to other pathogenic CoVs that cause SARS 74 and MERS. 75 Main Text 76 Introduction 77 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 78 2019 to cause a global pandemic of coronavirus disease 2019 (COVID-19) (1). SARS-CoV-2 79 causes a respiratory infection with acute respiratory distress syndrome (ARDS) in severe cases. 80 Innate antiviral responses, which include type I interferons (IFNs) are the first line of defense 81 after a virus enters a cell (2). Cellular pattern recognition receptors (PRRs) recognize viral 82 nucleic acids and activate key cellular kinases, such as inhibitor of nuclear factor kappa-B kinase 83 subunit epsilon (IKKe) and TANK-binding kinase 1 (TBK1). These kinases activate 84 transcription factors, such as interferon regulatory factor 3 (IRF3) to stimulate downstream 85 production of type I IFNs (3). 86 To counteract host antiviral responses, viruses encode proteins that can modulate type I 87 IFN production and signaling (4, 5). Emerging pathogenic human coronaviruses, such as SARS- 88 CoV and Middle East respiratory syndrome (MERS)-CoV have evolved multiple proteins that 89 can inhibit type I IFN responses in human cells (6-10). Thus, to better understand SARS-CoV-2 90 pathogenesis, it is critical to identify the early dynamic interactions of SARS-CoV-2 and the type 91 I IFN response. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.158154; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 92 Data from in vitro and in vivo work have demonstrated the lack of induction of type I IFN 93 responses following SARS-CoV-2 infection (11). Interestingly, on the contrary, emerging data 94 from patients with mild and moderate cases of COVID-19 have demonstrated the presence of 95 type I IFN (12, 13). Thus, the inability to mount an effective IFN response to SARS-CoV-2 may 96 also be associated with underlying host factors, along with the duration and extent of viral 97 infection. Furthermore, it is unclear if SARS-CoV-2 is unable to stimulate a type I IFN response 98 or actively suppresses the response after initiating it in infected cells. 99 In this study, we have identified global early transcriptional responses that are initiated 100 during SARS-CoV-2 infection of human lung epithelial (Calu-3) cells at 0, 1, 2, 3, 6, and 12 101 hours post infection. SARS-CoV-2 infected cells mounted a type I IFN response between 6 and 102 12 hours post infection (hpi) and the degree of this response correlated with virus replication and 103 transcription. However, a high dose infection of SARS-CoV-2 is unable to modulate poly (I:C)- 104 induced IFNβ production and signaling. Furthermore, SARS-CoV-2 is unable to modulate 105 interferon stimulated gene (ISG) expression in response to exogenous IFNβ. Our study provides 106 insights into early host responses that are generated on infection with SARS-CoV-2 and the 107 inability of the virus to efficiently modulate these responses, which may explain the low case- 108 fatality rate of COVID-19. Furthermore, it is likely that comorbidities and deficiencies in type I 109 IFN responses are associated with severe outcomes in COVID-19 patients. In summary, our data 110 indicate that SARS-CoV-2 is inefficient in modulating type I IFN production and signaling when 111 cells are exogenously stimulated. Further investigations into the ability of SARS-CoV-2 to mask 112 its nucleic acid pathogen associated molecular pattern (PAMP) from cellular PRRs to generate a 113 dampened innate antiviral response is warranted. 114 Results 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.158154; this version posted June 18, 2020.
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