1887

Abstract

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.052910-0
2013-08-01
2024-10-03
Loading full text...

Full text loading...

/deliver/fulltext/jgv/94/8/1749.html?itemId=/content/journal/jgv/10.1099/vir.0.052910-0&mimeType=html&fmt=ahah

References

  1. Belouzard S., Millet J. K., Licitra B. N., Whittaker G. R. 2012; Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4:1011–1033 [View Article][PubMed]
    [Google Scholar]
  2. Bergman S. J., Ferguson M. C., Santanello C. 2011; Interferons as therapeutic agents for infectious diseases. Infect Dis Clin North Am 25:819–834 [View Article][PubMed]
    [Google Scholar]
  3. Brockway S. M., Clay C. T., Lu X. T., Denison M. R. 2003; Characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase. J Virol 77:10515–10527 [View Article][PubMed]
    [Google Scholar]
  4. Cameron M. J., Kelvin A. A., Leon A. J., Cameron C. M., Ran L., Xu L., Chu Y. K., Danesh A., Fang Y. other authors 2012; Lack of innate interferon responses during SARS coronavirus infection in a vaccination and reinfection ferret model. PLoS ONE 7:e45842 [View Article][PubMed]
    [Google Scholar]
  5. Cervantes-Barragan L., Züst R., Weber F., Spiegel M., Lang K. S., Akira S., Thiel V., Ludewig B. 2007; Control of coronavirus infection through plasmacytoid dendritic-cell-derived type I interferon. Blood 109:1131–1137 [View Article][PubMed]
    [Google Scholar]
  6. de Groot R. J., Cowley J. A., Enjuanes L., Faaberg K. S., Perlman S., Rottier P. J., Snijder E. J., Ziebuhr J., Gorbalenya A. E. 2012; Order of Nidovirales . In Virus Taxonomy, the 9th Report of the International Committee on Taxonomy of Viruses pp. 785–795 Edited by King A., Adams M., Carstens E., Lefkowitz E. J. Amsterdam: Academic Press;
    [Google Scholar]
  7. de Groot R. J., Baker S. C., Baric R. S., Brown C. S., Drosten C., Enjuanes L., Fouchier R. A., Galiano M., Gorbalenya A. E., other authors. 2013; Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group. J Virol 87: in press (doi: [View Article][PubMed]
    [Google Scholar]
  8. de Wilde A. H., Zevenhoven-Dobbe J. C., van der Meer Y., Thiel V., Narayanan K., Makino S., Snijder E. J., van Hemert M. J. 2011; Cyclosporin A inhibits the replication of diverse coronaviruses. J Gen Virol 92:2542–2548 [View Article][PubMed]
    [Google Scholar]
  9. Drosten C., Günther S., Preiser W., van der Werf S., Brodt H. R., Becker S., Rabenau H., Panning M., Kolesnikova L. other authors 2003; Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976 [View Article][PubMed]
    [Google Scholar]
  10. Fouchier R. A., Hartwig N. G., Bestebroer T. M., Niemeyer B., de Jong J. C., Simon J. H., Osterhaus A. D. 2004; A previously undescribed coronavirus associated with respiratory disease in humans. Proc Natl Acad Sci U S A 101:6212–6216 [View Article][PubMed]
    [Google Scholar]
  11. Frieman M., Yount B., Heise M., Kopecky-Bromberg S. A., Palese P., Baric R. S. 2007; Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane. J Virol 81:9812–9824 [View Article][PubMed]
    [Google Scholar]
  12. Garlinghouse L. E. Jr, Smith A. L., Holford T. 1984; The biological relationship of mouse hepatitis virus (MHV) strains and interferon: in vitro induction and sensitivities. Arch Virol 82:19–29 [View Article][PubMed]
    [Google Scholar]
  13. Gorbalenya A. E., Enjuanes L., Ziebuhr J., Snijder E. J. 2006; Nidovirales: evolving the largest RNA virus genome. Virus Res 117:17–37 [View Article][PubMed]
    [Google Scholar]
  14. Gosert R., Kanjanahaluethai A., Egger D., Bienz K., Baker S. C. 2002; RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol 76:3697–3708 [View Article][PubMed]
    [Google Scholar]
  15. Haagmans B. L., Kuiken T., Martina B. E., Fouchier R. A., Rimmelzwaan G. F., van Amerongen G., van Riel D., de Jong T., Itamura S. other authors 2004; Pegylated interferon-α protects type 1 pneumocytes against SARS coronavirus infection in macaques. Nat Med 10:290–293 [View Article][PubMed]
    [Google Scholar]
  16. Hamre D., Procknow J. J. 1966; A new virus isolated from the human respiratory tract. Proc Soc Exp Biol Med 121:190–193[PubMed] [CrossRef]
    [Google Scholar]
  17. Hussain S., Perlman S., Gallagher T. M. 2008; Severe acute respiratory syndrome coronavirus protein 6 accelerates murine hepatitis virus infections by more than one mechanism. J Virol 82:7212–7222 [View Article][PubMed]
    [Google Scholar]
  18. Huynh J., Li S., Yount B., Smith A., Sturges L., Olsen J. C., Nagel J., Johnson J. B., Agnihothram S. other authors 2012; Evidence supporting a zoonotic origin of human coronavirus strain NL63. J Virol 86:12816–12825 [View Article][PubMed]
    [Google Scholar]
  19. Kindler E., Jónsdóttir H. R., Muth D., Hamming O. J., Hartmann R., Rodriguez R., Geffers R., Fouchier R. A., Drosten C. other authors 2013; Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential. MBio 4:e00611-12 [View Article][PubMed]
    [Google Scholar]
  20. Knoops K., Kikkert M., Worm S. H., Zevenhoven-Dobbe J. C., van der Meer Y., Koster A. J., Mommaas A. M., Snijder E. J. 2008; SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6:e226 [View Article][PubMed]
    [Google Scholar]
  21. Knoops K., Bárcena M., Limpens R. W., Koster A. J., Mommaas A. M., Snijder E. J. 2012; Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis. J Virol 86:2474–2487 [View Article][PubMed]
    [Google Scholar]
  22. Kopecky-Bromberg S. A., Martinez-Sobrido L., Palese P. 2006; 7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase. J Virol 80:785–793 [View Article][PubMed]
    [Google Scholar]
  23. Ksiazek T. G., Erdman D., Goldsmith C. S., Zaki S. R., Peret T., Emery S., Tong S., Urbani C., Comer J. A. other authors 2003; A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966 [View Article][PubMed]
    [Google Scholar]
  24. Kuiken T., Fouchier R. A., Schutten M., Rimmelzwaan G. F., van Amerongen G., van Riel D., Laman J. D., de Jong T., van Doornum G. other authors 2003; Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362:263–270 [View Article][PubMed]
    [Google Scholar]
  25. Lau S. K., Woo P. C., Li K. S., Huang Y., Tsoi H. W., Wong B. H., Wong S. S., Leung S. Y., Chan K. H., Yuen K. Y. 2005; Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102:14040–14045 [View Article][PubMed]
    [Google Scholar]
  26. Li W., Shi Z., Yu M., Ren W., Smith C., Epstein J. H., Wang H., Crameri G., Hu Z. other authors 2005; Bats are natural reservoirs of SARS-like coronaviruses. Science 310:676–679 [View Article][PubMed]
    [Google Scholar]
  27. McIntosh K., Dees J. H., Becker W. B., Kapikian A. Z., Chanock R. M. 1967; Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Proc Natl Acad Sci U S A 57:933–940 [View Article][PubMed]
    [Google Scholar]
  28. Müller M. A., Raj V. S., Muth D., Meyer B., Kallies S., Smits S. L., Wollny R., Bestebroer T. M., Specht S. other authors 2012; Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines. MBio 3:e00515-12 [View Article][PubMed]
    [Google Scholar]
  29. Nagy P. D., Wang R. Y., Pogany J., Hafren A., Makinen K. 2011; Emerging picture of host chaperone and cyclophilin roles in RNA virus replication. Virology 411:374–382 [View Article][PubMed]
    [Google Scholar]
  30. Paragas J., Blatt L. M., Hartmann C., Huggins J. W., Endy T. P. 2005; Interferon alfacon1 is an inhibitor of SARS-corona virus in cell-based models. Antiviral Res 66:99–102 [View Article][PubMed]
    [Google Scholar]
  31. Perlman S., Netland J. 2009; Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7:439–450 [View Article][PubMed]
    [Google Scholar]
  32. Pfefferle S., Oppong S., Drexler J. F., Gloza-Rausch F., Ipsen A., Seebens A., Müller M. A., Annan A., Vallo P. other authors 2009; Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana. Emerg Infect Dis 15:1377–1384 [View Article][PubMed]
    [Google Scholar]
  33. Pfefferle S., Schöpf J., Kögl M., Friedel C. C., Müller M. A., Carbajo-Lozoya J., Stellberger T., von Dall’Armi E., Herzog P. other authors 2011; The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7:e1002331 [View Article][PubMed]
    [Google Scholar]
  34. Raj V. S., Mou H., Smits S. L., Dekkers D. H., Müller M. A., Dijkman R., Muth D., Demmers J. A., Zaki A. other authors 2013; Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495:251–254 [View Article][PubMed]
    [Google Scholar]
  35. Randall R. E., Goodbourn S. 2008; Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 89:1–47 [View Article][PubMed]
    [Google Scholar]
  36. Ratia K., Saikatendu K. S., Santarsiero B. D., Barretto N., Baker S. C., Stevens R. C., Mesecar A. D. 2006; Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proc Natl Acad Sci U S A 103:5717–5722 [View Article][PubMed]
    [Google Scholar]
  37. Rose K. M., Elliott R., Martínez-Sobrido L., García-Sastre A., Weiss S. R. 2010; Murine coronavirus delays expression of a subset of interferon-stimulated genes. J Virol 84:5656–5669 [View Article][PubMed]
    [Google Scholar]
  38. Roth-Cross J. K., Martínez-Sobrido L., Scott E. P., García-Sastre A., Weiss S. R. 2007; Inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels. J Virol 81:7189–7199 [View Article][PubMed]
    [Google Scholar]
  39. Sims A. C., Tilton S. C., Menachery V. D., Gralinski L. E., Schäfer A., Matzke M. M., Webb-Robertson B. J., Chang J., Luna M. L. other authors 2013; Release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells. J Virol 87:3885–3902 [View Article][PubMed]
    [Google Scholar]
  40. Snijder E. J., Wassenaar A. L., Spaan W. J. 1994; Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J Virol 68:5755–5764[PubMed]
    [Google Scholar]
  41. Snijder E. J., Bredenbeek P. J., Dobbe J. C., Thiel V., Ziebuhr J., Poon L. L., Guan Y., Rozanov M., Spaan W. J., Gorbalenya A. E. 2003; Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331:991–1004 [View Article][PubMed]
    [Google Scholar]
  42. Snijder E. J., van der Meer Y., Zevenhoven-Dobbe J., Onderwater J. J., van der Meulen J., Koerten H. K., Mommaas A. M. 2006; Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol 80:5927–5940 [View Article][PubMed]
    [Google Scholar]
  43. Stertz S., Reichelt M., Spiegel M., Kuri T., Martínez-Sobrido L., García-Sastre A., Weber F., Kochs G. 2007; The intracellular sites of early replication and budding of SARS-coronavirus. Virology 361:304–315 [View Article][PubMed]
    [Google Scholar]
  44. Taguchi F., Siddell S. G. 1985; Difference in sensitivity to interferon among mouse hepatitis viruses with high and low virulence for mice. Virology 147:41–48 [View Article][PubMed]
    [Google Scholar]
  45. Ulasli M., Verheije M. H., de Haan C. A., Reggiori F. 2010; Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus. Cell Microbiol 12:844–861 [View Article][PubMed]
    [Google Scholar]
  46. van Boheemen S., de Graaf M., Lauber C., Bestebroer T. M., Raj V. S., Zaki A. M., Osterhaus A. D., Haagmans B. L., Gorbalenya A. E. other authors 2012; Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. MBio 3:e00473-12 [View Article][PubMed]
    [Google Scholar]
  47. van den Worm S. H., Eriksson K. K., Zevenhoven J. C., Weber F., Züst R., Kuri T., Dijkman R., Chang G., Siddell S. G. other authors 2012; Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. PLoS ONE 7:e32857 [View Article][PubMed]
    [Google Scholar]
  48. van der Hoek L., Pyrc K., Jebbink M. F., Vermeulen-Oost W., Berkhout R. J., Wolthers K. C., Wertheim-van Dillen P. M., Kaandorp J., Spaargaren J., Berkhout B. 2004; Identification of a new human coronavirus. Nat Med 10:368–373 [View Article][PubMed]
    [Google Scholar]
  49. van der Meer Y., van Tol H., Locker J. K., Snijder E. J. 1998; ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex. J Virol 72:6689–6698[PubMed]
    [Google Scholar]
  50. van Hemert M. J., de Wilde A. H., Gorbalenya A. E., Snijder E. J. 2008a; The in vitro RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor. J Biol Chem 283:16525–16536 [View Article][PubMed]
    [Google Scholar]
  51. van Hemert M. J., van den Worm S. H., Knoops K., Mommaas A. M., Gorbalenya A. E., Snijder E. J. 2008b; SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro . PLoS Pathog 4:e1000054 [View Article][PubMed]
    [Google Scholar]
  52. van Kasteren P. B., Bailey-Elkin B. A., James T. W., Ninaber D. K., Beugeling C., Khajehpour M., Snijder E. J., Mark B. L., Kikkert M. 2013; Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells. Proc Natl Acad Sci U S A 110:E838–E847 [View Article][PubMed]
    [Google Scholar]
  53. Versteeg G. A., Bredenbeek P. J., van den Worm S. H., Spaan W. J. 2007; Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition. Virology 361:18–26 [View Article][PubMed]
    [Google Scholar]
  54. Vijaykrishna D., Smith G. J., Zhang J. X., Peiris J. S., Chen H., Guan Y. 2007; Evolutionary insights into the ecology of coronaviruses. J Virol 81:4012–4020 [View Article][PubMed]
    [Google Scholar]
  55. Vijgen L., Keyaerts E., Moës E., Thoelen I., Wollants E., Lemey P., Vandamme A. M., Van Ranst M. 2005; Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event. J Virol 79:1595–1604 [View Article][PubMed]
    [Google Scholar]
  56. Weber F., Wagner V., Rasmussen S. B., Hartmann R., Paludan S. R. 2006; Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 80:5059–5064 [View Article][PubMed]
    [Google Scholar]
  57. Woo P. C., Lau S. K., Chu C. M., Chan K. H., Tsoi H. W., Huang Y., Wong B. H., Poon R. W., Cai J. J. other authors 2005; Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J Virol 79:884–895 [View Article][PubMed]
    [Google Scholar]
  58. Woo P. C., Wang M., Lau S. K., Xu H., Poon R. W., Guo R., Wong B. H., Gao K., Tsoi H. W. other authors 2007; Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features. J Virol 81:1574–1585 [View Article][PubMed]
    [Google Scholar]
  59. Yoshikawa T., Hill T. E., Yoshikawa N., Popov V. L., Galindo C. L., Garner H. R., Peters C. J., Tseng C. T. 2010; Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection. PLoS ONE 5:e8729 [View Article][PubMed]
    [Google Scholar]
  60. Zaki A. M., van Boheemen S., Bestebroer T. M., Osterhaus A. D., Fouchier R. A. 2012; Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820 [View Article][PubMed]
    [Google Scholar]
  61. Zeng F. Y., Chan C. W., Chan M. N., Chen J. D., Chow K. Y., Hon C. C., Hui K. H., Li J., Li V. Y. other authors 2003; The complete genome sequence of severe acute respiratory syndrome coronavirus strain HKU-39849 (HK-39). Exp Biol Med (Maywood) 228:866–873[PubMed]
    [Google Scholar]
  62. Zheng B., He M. L., Wong K. L., Lum C. T., Poon L. L., Peng Y., Guan Y., Lin M. C., Kung H. F. 2004; Potent inhibition of SARS-associated coronavirus (SCOV) infection and replication by type I interferons (IFN-α/β) but not by type II interferon (IFN-γ). J Interferon Cytokine Res 24:388–390 [View Article][PubMed]
    [Google Scholar]
  63. Zhong Y., Tan Y. W., Liu D. X. 2012; Recent progress in studies of arterivirus– and coronavirus–host interactions. Viruses 4:980–1010 [View Article][PubMed]
    [Google Scholar]
  64. Zhou P., Li H., Wang H., Wang L. F., Shi Z. 2012; Bat severe acute respiratory syndrome-like coronavirus ORF3b homologues display different interferon antagonist activities. J Gen Virol 93:275–281 [View Article][PubMed]
    [Google Scholar]
  65. Züst R., Cervantes-Barragan L., Habjan M., Maier R., Neuman B. W., Ziebuhr J., Szretter K. J., Baker S. C., Barchet W. other authors 2011; Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat Immunol 12:137–143 [View Article][PubMed]
    [Google Scholar]
/content/journal/jgv/10.1099/vir.0.052910-0
Loading
/content/journal/jgv/10.1099/vir.0.052910-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error