Review Article | Published:

ISG15 in antiviral immunity and beyond

Nature Reviews Microbiologyvolume 16pages423439 (2018) | Download Citation


The host response to viral infection includes the induction of type I interferons and the subsequent upregulation of hundreds of interferon-stimulated genes. Ubiquitin-like protein ISG15 is an interferon-induced protein that has been implicated as a central player in the host antiviral response. Over the past 15 years, efforts to understand how ISG15 protects the host during infection have revealed that its actions are diverse and pathogen-dependent. In this Review, we describe new insights into how ISG15 directly inhibits viral replication and discuss the recent finding that ISG15 modulates the host damage and repair response, immune response and other host signalling pathways. We also explore the viral immune-evasion strategies that counteract the actions of ISG15. These findings are integrated with a discussion of the recent identification of ISG15-deficient individuals and a cellular receptor for ISG15 that provides new insights into how ISG15 shapes the host response to viral infection.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Schneider, W. M., Chevillotte, M. D. & Rice, C. M. Interferon-stimulated genes: a complex web of host defenses. Annu. Rev. Immunol. 32, 513–545 (2014).

  2. 2.

    Der, S. D., Zhou, A., Williams, B. R. & Silverman, R. H. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc. Natl Acad. Sci. USA 95, 15623–15628 (1998).

  3. 3.

    Loeb, K. R. & Haas, A. L. The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. J. Biol. Chem. 267, 7806–7813 (1992).

  4. 4.

    Zhang, X. et al. Human intracellular ISG15 prevents interferon-alpha/beta over-amplification and auto-inflammation. Nature 517, 89–93 (2015). This study identifies a second cohort of individuals lacking ISG15 who presented with evidence of interferon hypersensitivity. They demonstrated that human ISG15 non-covalently binds to USP18, preventing its ubiquitylation and subsequent degradation, and therefore functions as a key negative regulator of type I interferon signalling.

  5. 5.

    Bogunovic, D. et al. Mycobacterial disease and impaired IFN-gamma immunity in humans with inherited ISG15 deficiency. Science 337, 1684–1688 (2012). This groundbreaking study reports the first ISG15-deficient individuals. The findings indicate that these patients developed disseminated mycobacterial disease after bacillus Calmette–Guérin (BCG) vaccination and reveal that cells derived from these patients produced reduced levels of IFNγ after stimulation with Mycobacterium owing to the loss of extracellular ISG15 and its ability to function as a cytokine to stimulate IFNγ production.

  6. 6.

    Korant, B. D., Blomstrom, D. C., Jonak, G. J. & Knight, E. Jr. Interferon-induced proteins. Purification and characterization of a 15,000-dalton protein from human and bovine cells induced by interferon. J. Biol. Chem. 259, 14835–14839 (1984).

  7. 7.

    Haas, A. L., Ahrens, P., Bright, P. M. & Ankel, H. Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin. J. Biol. Chem. 262, 11315–11323 (1987).

  8. 8.

    Blomstrom, D. C., Fahey, D., Kutny, R., Korant, B. D. & Knight, E. Jr. Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence. J. Biol. Chem. 261, 8811–8816 (1986).

  9. 9.

    Dao, C. T. & Zhang, D. E. ISG15: a ubiquitin-like enigma. Front. Biosci. 10, 2701–2722 (2005).

  10. 10.

    Narasimhan, J. et al. Crystal structure of the interferon-induced ubiquitin-like protein ISG15. J. Biol. Chem. 280, 27356–27365 (2005).

  11. 11.

    Yuan, W. & Krug, R. M. Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J. 20, 362–371 (2001).

  12. 12.

    Radoshevich, L. et al. ISG15 counteracts Listeria monocytogenes infection. eLife 4, e06848 (2015).

  13. 13.

    Malakhova, O., Malakhov, M., Hetherington, C. & Zhang, D. E. Lipopolysaccharide activates the expression of ISG15-specific protease UBP43 via interferon regulatory factor 3. J. Biol. Chem. 277, 14703–14711 (2002).

  14. 14.

    Pitha-Rowe, I., Hassel, B. A. & Dmitrovsky, E. Involvement of UBE1L in ISG15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia. J. Biol. Chem. 279, 18178–18187 (2004).

  15. 15.

    Liu, M., Hummer, B. T., Li, X. & Hassel, B. A. Camptothecin induces the ubiquitin-like protein, ISG15, and enhances ISG15 conjugation in response to interferon. J. Interferon Cytokine Res. 24, 647–654 (2004).

  16. 16.

    Potter, J. L., Narasimhan, J., Mende-Mueller, L. & Haas, A. L. Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein. J. Biol. Chem. 274, 25061–25068 (1999).

  17. 17.

    Zhang, D. & Zhang, D. E. Interferon-stimulated gene 15 and the protein ISGylation system. J. Interferon Cytokine Res. 31, 119–130 (2011).

  18. 18.

    Giannakopoulos, N. V. et al. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res. Commun. 336, 496–506 (2005).

  19. 19.

    Zhao, C., Denison, C., Huibregtse, J. M., Gygi, S. & Krug, R. M. Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc. Natl Acad. Sci. USA 102, 10200–10205 (2005).

  20. 20.

    Liu, M., Li, X. L. & Hassel, B. A. Proteasomes modulate conjugation to the ubiquitin-like protein, ISG15. J. Biol. Chem. 278, 1594–1602 (2003).

  21. 21.

    Desai, S. D. et al. Elevated expression of ISG15 in tumor cells interferes with the ubiquitin/26S proteasome pathway. Cancer Res. 66, 921–928 (2006).

  22. 22.

    Fan, J. B. et al. Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis. Sci. Rep. 5, 12704 (2015).

  23. 23.

    Jeon, Y. J. et al. ISG15 modification of filamin B negatively regulates the type I interferon-induced JNK signalling pathway. EMBO Rep. 10, 374–380 (2009).

  24. 24.

    Malakhov, M. P., Malakhova, O. A., Kim, K. I., Ritchie, K. J. & Zhang, D. E. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J. Biol. Chem. 277, 9976–9981 (2002).

  25. 25.

    Basters, A. et al. Structural basis of the specificity of USP18 toward ISG15. Nat. Struct. Mol. Biol. 24, 270–278 (2017). This structural-based study characterizes how USP18 specifically recognizes and deconjugates ISG15-conjugated proteins.

  26. 26.

    Malakhova, O. A. et al. UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity. EMBO J. 25, 2358–2367 (2006). This was the first paper to demonstrate that, in addition to functioning as a deISGylase, USP18 also binds to the type I interferon receptor and functions as a critical negative regulator of interferon signalling.

  27. 27.

    Knobeloch, K. P., Utermohlen, O., Kisser, A., Prinz, M. & Horak, I. Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice. Mol. Cell. Biol. 25, 11030–11034 (2005).

  28. 28.

    Knight, E. Jr & Cordova, B. IFN-induced 15-kDa protein is released from human lymphocytes and monocytes. J. Immunol. 146, 2280–2284 (1991).

  29. 29.

    D’Cunha, J. et al. In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine. J. Immunol. 157, 4100–4108 (1996).

  30. 30.

    Lai, C. et al. Mice lacking the ISG15 E1 enzyme UbE1L demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza B virus infection. J. Virol. 83, 1147–1151 (2009).

  31. 31.

    Werneke, S. W. et al. ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation. PLoS Pathog. 7, e1002322 (2011). This is the first in vivo study to indicate that unconjugated ISG15 can protect the host from viral infection by functioning as a critical immunomodulatory molecule.

  32. 32.

    D’Cunha, J. et al. Immunoregulatory properties of ISG15, an interferon-induced cytokine. Proc. Natl Acad. Sci. USA 93, 211–215 (1996).

  33. 33.

    Padovan, E. et al. Interferon stimulated gene 15 constitutively produced by melanoma cells induces e-cadherin expression on human dendritic cells. Cancer Res. 62, 3453–3458 (2002).

  34. 34.

    Owhashi, M. et al. Identification of a ubiquitin family protein as a novel neutrophil chemotactic factor. Biochem. Biophys. Res. Commun. 309, 533–539 (2003).

  35. 35.

    Sun, L. et al. Exosomes contribute to the transmission of anti-HIV activity from TLR3-activated brain microvascular endothelial cells to macrophages. Antiviral Res. 134, 167–171 (2016).

  36. 36.

    Dos Santos, P. F. & Mansur, D. S. Beyond ISGlylation: functions of free intracellular and extracellular ISG15. J. Interferon Cytokine Res. 37, 246–253 (2017).

  37. 37.

    Swaim, C. D., Scott, A. F., Canadeo, L. A. & Huibregtse, J. M. Extracellular ISG15 signals cytokine secretion through the LFA-1 integrin receptor. Mol. Cell 68, 581–590.e5 (2017). This paper identifies the first cell surface receptor for ISG15 and demonstrates its ability to augment IFNγ secretion from cells that were primed with IL-12.

  38. 38.

    Narasimhan, J., Potter, J. L. & Haas, A. L. Conjugation of the 15-kDa interferon-induced ubiquitin homolog is distinct from that of ubiquitin. J. Biol. Chem. 271, 324–330 (1996).

  39. 39.

    Okumura, A., Pitha, P. M. & Harty, R. N. ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc. Natl Acad. Sci. USA 105, 3974–3979 (2008). This mechanistic study shows that ISG15 inhibits viral budding by targeting the E3 ligase activity of NEDD4.

  40. 40.

    Nakashima, H., Nguyen, T., Goins, W. F. & Chiocca, E. A. Interferon-stimulated gene 15 (ISG15) and ISG15-linked proteins can associate with members of the selective autophagic process, histone deacetylase 6 (HDAC6) and SQSTM1/p62. J. Biol. Chem. 290, 1485–1495 (2015).

  41. 41.

    Du, Y. et al. LRRC25 inhibits type I IFN signaling by targeting ISG15-associated RIG-I for autophagic degradation. EMBO J. 37, 351–366 (2018).

  42. 42.

    Lenschow, D. J. et al. Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo. J. Virol. 79, 13974–13983 (2005).

  43. 43.

    Sampson, D. L. et al. A four-biomarker blood signature discriminates systemic inflammation due to viral infection versus other etiologies. Sci. Rep. 7, 2914 (2017).

  44. 44.

    Morales, D. J. & Lenschow, D. J. The antiviral activities of ISG15. J. Mol. Biol. 425, 4995–5008 (2013).

  45. 45.

    Hermann, M. & Bogunovic, D. ISG15: in sickness and in health. Trends Immunol. 38, 79–93 (2017).

  46. 46.

    Giannakopoulos, N. V. et al. ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus. J. Virol. 83, 1602–1610 (2009).

  47. 47.

    Lenschow, D. J. et al. IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses. Proc. Natl Acad. Sci. USA 104, 1371–1376 (2007). This is the first in vivo study of ISG15-deficient mice, which demonstrates that ISG15 protected mice from viral-induced lethality and that it is critical in the host response to viral infection.

  48. 48.

    Rahnefeld, A. et al. Ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy. Circulation 130, 1589–1600 (2014). This paper indicates that ISG15 conjugation has a critical role in controlling CVB3 viral replication and viral-induced cardiomyopathy.

  49. 49.

    Ketscher, L. et al. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc. Natl Acad. Sci. USA 112, 1577–1582 (2015). The authors generate Usp18 -knock-in mice in which USP18 is mutated so it cannot function as a deISGylase but still maintains its ability to negatively regulate interferon signalling. Analysis of these mice reveals that an increase in ISGylation could mediate viral resistance.

  50. 50.

    Morales, D. J. et al. Novel mode of ISG15-mediated protection against influenza A virus and Sendai virus in mice. J. Virol. 89, 337–349 (2015). This paper describes the ability of ISG15 to protect the host from viral-induced lethality, independent of its effects on viral replication (a process defined as disease tolerance).

  51. 51.

    Xu, D. et al. Modification of BECN1 by ISG15 plays a crucial role in autophagy regulation by type I IFN/interferon. Autophagy 11, 617–628 (2015).

  52. 52.

    Baldanta, S. et al. ISG15 governs mitochondrial function in macrophages following vaccinia virus infection. PLoS Pathog. 13, e1006651 (2017).

  53. 53.

    Speer, S. D. et al. ISG15 deficiency and increased viral resistance in humans but not mice. Nat. Commun. 7, 11496 (2016). This paper describes differences in viral resistance between human and mouse cells lacking ISG15 and demonstrates that this is due to the differential ability of human and mouse ISG15 to negatively regulate IFNα and IFNβ signalling.

  54. 54.

    Durfee, L. A., Lyon, N., Seo, K. & Huibregtse, J. M. The ISG15 conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of ISG15. Mol. Cell 38, 722–732 (2010). This study provides the first evidence that ISG15 conjugation targets newly synthesized proteins. It demonstrates that overexpression of most proteins, along with the ISG15 conjugation cascade, can lead to their modification.

  55. 55.

    Zhao, C., Hsiang, T. Y., Kuo, R. L. & Krug, R. M. ISG15 conjugation system targets the viral NS1 protein in influenza A virus-infected cells. Proc. Natl Acad. Sci. USA 107, 2253–2258 (2010).

  56. 56.

    Tang, Y. et al. Herc5 attenuates influenza A virus by catalyzing ISGylation of viral NS1 protein. J. Immunol. 184, 5777–5790 (2010). Together with reference 55, this is one of the first studies to describe that a viral protein (IAV NS1) can be ISGylated.

  57. 57.

    Wang, X. et al. Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon. J. Virol. 74, 11566–11573 (2000).

  58. 58.

    Bergmann, M. et al. Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J. Virol. 74, 6203–6206 (2000).

  59. 59.

    de la Luna, S., Fortes, P., Beloso, A. & Ortin, J. Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J. Virol. 69, 2427–2433 (1995).

  60. 60.

    Nemeroff, M. E., Barabino, S. M., Li, Y., Keller, W. & Krug, R. M. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3’end formation of cellular pre-mRNAs. Mol. Cell 1, 991–1000 (1998).

  61. 61.

    Fortes, P., Beloso, A. & Ortin, J. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 13, 704–712 (1994).

  62. 62.

    Zhao, C. et al. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins. Nat. Commun. 7, 12754 (2016). This study finds that NS1/B binds to and sequesters ISGylated viral proteins, particularly ISGylated viral NPs, which prevents the incorporation of ISGylated NPs into NP oligomers, which was previously shown to inhibit viral RNA synthesis and viral replication.

  63. 63.

    Mathers, C., Schafer, X., Martinez-Sobrido, L. & Munger, J. The human cytomegalovirus UL26 protein antagonizes NF-kappaB activation. J. Virol. 88, 14289–14300 (2014).

  64. 64.

    Kim, Y. J. et al. Consecutive inhibition of ISG15 expression and ISGylation by cytomegalovirus regulators. PLoS Pathog. 12, e1005850 (2016). This paper identifies HCMV viral proteins that antagonize the ISG15 pathway to facilitate viral infection.

  65. 65.

    Okumura, A., Lu, G., Pitha-Rowe, I. & Pitha, P. M. Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15. Proc. Natl Acad. Sci. USA 103, 1440–1445 (2006).

  66. 66.

    Sanyal, S. et al. Type I interferon imposes a TSG101/ISG15 checkpoint at the Golgi for glycoprotein trafficking during influenza virus infection. Cell Host Microbe 14, 510–521 (2013). This mechanistic study finds that ISGylation of host protein in the secretory pathway impedes influenza virus release.

  67. 67.

    Villarroya-Beltri, C. et al. ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins. Nat. Commun. 7, 13588 (2016).

  68. 68.

    Yasuda, J., Nakao, M., Kawaoka, Y. & Shida, H. Nedd4 regulates egress of Ebola virus-like particles from host cells. J. Virol. 77, 9987–9992 (2003).

  69. 69.

    Malakhova, O. A. & Zhang, D. E. ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response. J. Biol. Chem. 283, 8783–8787 (2008).

  70. 70.

    Han, Z. et al. ITCH E3 ubiquitin ligase interacts with Ebola virus VP40 to regulate budding. J. Virol. 90, 9163–9171 (2016).

  71. 71.

    Pincetic, A. & Leis, J. The mechanism of budding of retroviruses from cell membranes. Adv. Virol. 2009, 6239691–6239699 (2009).

  72. 72.

    Pincetic, A., Kuang, Z., Seo, E. J. & Leis, J. The interferon-induced gene ISG15 blocks retrovirus release from cells late in the budding process. J. Virol. 84, 4725–4736 (2010).

  73. 73.

    Kuang, Z., Seo, E. J. & Leis, J. Mechanism of inhibition of retrovirus release from cells by interferon-induced gene ISG15. J. Virol. 85, 7153–7161 (2011).

  74. 74.

    Dai, L. et al. Transcriptomic analysis of KSHV-infected primary oral fibroblasts: the role of interferon-induced genes in the latency of oncogenic virus. Oncotarget 7, 47052–47060 (2016).

  75. 75.

    Jacobs, S. R. et al. Kaposi’s sarcoma-associated herpesvirus viral interferon regulatory factor 1 interacts with a member of the interferon-stimulated gene 15 pathway. J. Virol. 89, 11572–11583 (2015). The study demonstrates that ISG15 regulates reactivation of latent virus.

  76. 76.

    Werneke, S. W. A. Role for Interferon Stimulated Gene-15 (ISG15) During Chikungunya Virus Infection Thesis, Washington Univ. (2013).

  77. 77.

    Eduardo-Correia, B., Martinez-Romero, C., Garcia-Sastre, A. & Guerra, S. ISG15 is counteracted by vaccinia virus E3 protein and controls the proinflammatory response against viral infection. J. Virol. 88, 2312–2318 (2014).

  78. 78.

    Soares, M. P., Teixeira, L. & Moita, L. F. Disease tolerance and immunity in host protection against infection. Nat. Rev. Immunol. 17, 83–96 (2017).

  79. 79.

    Shi, H. X. et al. Positive regulation of interferon regulatory factor 3 activation by Herc5 via ISG15 modification. Mol. Cell. Biol. 30, 2424–2436 (2010).

  80. 80.

    Katzenell, S. & Leib, D. A. Herpes simplex virus and interferon signaling induce novel autophagic clusters in sensory neurons. J. Virol. 90, 4706–4719 (2016).

  81. 81.

    Falvey, C. M. et al. UBE2L6/UBCH8 and ISG15 attenuate autophagy in esophageal cancer cells. Oncotarget 8, 23479–23491 (2017).

  82. 82.

    Malakhov, M. P. et al. High-throughput immunoblotting. Ubiquitiin-like protein ISG15 modifies key regulators of signal transduction. J. Biol. Chem. 278, 16608–16613 (2003).

  83. 83.

    Okumura, F. et al. Activation of double-stranded RNA-activated protein kinase (PKR) by interferon-stimulated gene 15 (ISG15) modification down-regulates protein translation. J. Biol. Chem. 288, 2839–2847 (2013).

  84. 84.

    Ganesan, M., Poluektova, L. Y., Tuma, D. J., Kharbanda, K. K. & Osna, N. A. Acetaldehyde disrupts interferon alpha signaling in hepatitis C virus-infected liver cells by up-regulating USP18. Alcohol Clin. Exp. Res. 40, 2329–2338 (2016).

  85. 85.

    Jones, D. M., Domingues, P., Targett-Adams, P. & McLauchlan, J. Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication. J. Gen. Virol. 91, 2238–2248 (2010).

  86. 86.

    Chen, L. et al. ISG15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis C virus production in vitro: implications for chronic infection and response to treatment. J. Gen. Virol. 91, 382–388 (2010).

  87. 87.

    Broering, R. et al. The interferon stimulated gene 15 functions as a proviral factor for the hepatitis C virus and as a regulator of the IFN response. Gut 59, 1111–1119 (2010).

  88. 88.

    Chua, P. K. et al. Modulation of alpha interferon anti-hepatitis C virus activity by ISG15. J. Gen. Virol. 90, 2929–2939 (2009).

  89. 89.

    Sung, P. S. et al. Roles of unphosphorylated ISGF3 in HCV infection and interferon responsiveness. Proc. Natl Acad. Sci. USA 112, 10443–10448 (2015). This study shows that ISG15 sustains USP18-mediated interferon signalling, which impedes the effectiveness of an HCV therapy.

  90. 90.

    Sridharan, H., Zhao, C. & Krug, R. M. Species specificity of the NS1 protein of influenza B virus: NS1 binds only human and non-human primate ubiquitin-like ISG15 proteins. J. Biol. Chem. 285, 7852–7856 (2010).

  91. 91.

    Versteeg, G. A. et al. Species-specific antagonism of host ISGylation by the influenza B virus NS1 protein. J. Virol. 84, 5423–5430 (2010).

  92. 92.

    Guerra, S., Caceres, A., Knobeloch, K. P., Horak, I. & Esteban, M. Vaccinia virus E3 protein prevents the antiviral action of ISG15. PLoS Pathog. 4, e1000096 (2008). The study shows that vaccinia virus E3L protein functions as an immune-evasion protein by inhibiting ISG15 conjugate formation and is critical to viral pathogenesis.

  93. 93.

    Frias-Staheli, N. et al. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe 2, 404–416 (2007). The study is the first to identify that viral OTU domain-containing proteins can function as both deubiquitinases and deISGylases.

  94. 94.

    Lindner, H. A. et al. The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme. J. Virol. 79, 15199–15208 (2005).

  95. 95.

    Clementz, M. A. et al. Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases. J. Virol. 84, 4619–4629 (2010).

  96. 96.

    Mielech, A. M., Kilianski, A., Baez-Santos, Y. M., Mesecar, A. D. & Baker, S. C. MERS-CoV papain-like protease has deISGylating and deubiquitinating activities. Virology 450–451, 64–70 (2014). The study demonstrates that SARS and MERS PLpro also function as deubiquitinating and deISGylating enzymes.

  97. 97.

    Chen, Z. et al. Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63. J. Virol. 81, 6007–6018 (2007).

  98. 98.

    Ma, X. Z. et al. Protein interferon-stimulated gene 15 conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis. J. Virol. 88, 6195–6204 (2014).

  99. 99.

    Deng, X. et al. A chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses. J. Virol. 88, 11825–11833 (2014). This study shows that the deISGylase activity of a SARS PLpro has a critical role during infection by targeting the ISG15 pathway.

  100. 100.

    Deaton, M. K. et al. Biochemical and structural insights into the preference of nairoviral DeISGylases for interferon-stimulated gene product 15 originating from certain species. J. Virol. 90, 8314–8327 (2016).

  101. 101.

    Bekes, M. et al. Recognition of Lys48-linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease. Mol. Cell 62, 572–585 (2016).

  102. 102.

    Altun, M. et al. The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages. PLoS ONE 10, e0115344 (2015).

  103. 103.

    Bekes, M. et al. SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme. Biochem. J. 468, 215–226 (2015).

  104. 104.

    Deaton, M. K., Spear, A., Faaberg, K. S. & Pegan, S. D. The vOTU domain of highly-pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference. Virology 454–455, 247–253 (2014).

  105. 105.

    Baez-Santos, Y. M., Mielech, A. M., Deng, X., Baker, S. & Mesecar, A. D. Catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the Middle East respiratory syndrome coronavirus. J. Virol. 88, 12511–12527 (2014).

  106. 106.

    Ratia, K., Kilianski, A., Baez-Santos, Y. M., Baker, S. C. & Mesecar, A. Structural basis for the ubiquitin-linkage specificity and deISGylating activity of SARS-CoV papain-like protease. PLoS Pathog. 10, e1004113 (2014).

  107. 107.

    Daczkowski, C. M. et al. Structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species. J. Mol. Biol. 429, 1661–1683 (2017).

  108. 108.

    Bianco, C. & Mohr, I. Restriction of human cytomegalovirus replication by ISG15, a host effector regulated by cGAS-STING double-stranded-DNA sensing. J. Virol. 91, e02483–16 (2017).

  109. 109.

    Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

  110. 110.

    Gane, E. J. et al. The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection. J. Hepatol. 63, 320–328 (2015).

  111. 111.

    Janssen, H. L. A. et al. Safety, efficacy and pharmacodynamics of vesatolimod (GS-9620) in virally-suppressed patients with chronic hepatitis B. J. Hepatol. 68, 431–440 (2018).

  112. 112.

    Villarreal, D. O. et al. Ubiquitin-like molecule ISG15 acts as an immune adjuvant to enhance antigen-specific CD8 T cell tumor immunity. Mol. Ther. 23, 1653–1662 (2015).

  113. 113.

    Malakhova, O. A. et al. Protein ISGylation modulates the JAK-STAT signaling pathway. Genes Dev. 17, 455–460 (2003).

  114. 114.

    Tokarz, S. et al. The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase. J. Biol. Chem. 279, 46424–46430 (2004).

  115. 115.

    Meuwissen, M. E. et al. Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J. Exp. Med. 213, 1163–1174 (2016).

  116. 116.

    Dauphinee, S. M. et al. Contribution of increased ISG15, ISGylation and deregulated type I IFN signaling in Usp18 mutant mice during the course of bacterial infections. Genes Immun. 15, 282–292 (2014).

  117. 117.

    Manca, C. et al. Hypervirulent M. tuberculosis W/Beijing strains upregulate type I IFNs and increase expression of negative regulators of the Jak-Stat pathway. J. Interferon Cytokine Res. 25, 694–701 (2005).

  118. 118.

    Ordway, D. et al. The hypervirulent Mycobacterium tuberculosis strain HN878 induces a potent TH1 response followed by rapid down-regulation. J. Immunol. 179, 522–531 (2007).

  119. 119.

    Stanley, S. A., Johndrow, J. E., Manzanillo, P. & Cox, J. S. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178, 3143–3152 (2007).

  120. 120.

    Antonelli, L. R. et al. Intranasal Poly-IC treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population. J. Clin. Invest. 120, 1674–1682 (2010).

  121. 121.

    Desvignes, L., Wolf, A. J. & Ernst, J. D. Dynamic roles of type I and type II IFNs in early infection with Mycobacterium tuberculosis. J. Immunol. 188, 6205–6215 (2012).

  122. 122.

    Dorhoi, A. et al. Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics. Eur. J. Immunol. 44, 2380–2393 (2014).

  123. 123.

    Kimmey, J. M. et al. The impact of ISGylation during Mycobacterium tuberculosis infection in mice. Microbes Infect. 19, 249–258 (2017).

  124. 124.

    Manca, C. et al. Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta. Proc. Natl Acad. Sci. USA 98, 5752–5757 (2001).

  125. 125.

    Berry, M. P. et al. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature 466, 973–977 (2010).

  126. 126.

    Dong, C., Gao, N., Ross, B. X. & Yu, F. X. ISG15 in host defense against Candida albicans infection in a mouse model of fungal keratitis. Invest. Ophthalmol. Vis. Sci. 58, 2948–2958 (2017).

  127. 127.

    Cheon, H. et al. IFNbeta-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage. EMBO J. 32, 2751–2763 (2013).

  128. 128.

    Cheon, H. & Stark, G. R. Unphosphorylated STAT1 prolongs the expression of interferon-induced immune regulatory genes. Proc. Natl Acad. Sci. USA 106, 9373–9378 (2009).

  129. 129.

    Kim, K. I. et al. Enhanced antibacterial potential in UBP43-deficient mice against Salmonella typhimurium infection by up-regulating type I IFN signaling. J. Immunol. 175, 847–854 (2005).

  130. 130.

    Dao, C. T., Luo, J. K. & Zhang, D. E. Retinoic acid-induced protein ISGylation is dependent on interferon signal transduction. Blood Cells Mol. Dis. 36, 406–413 (2006).

  131. 131.

    Memet, S., Besancon, F., Bourgeade, M. F. & Thang, M. N. Direct induction of interferon-gamma- and interferon-alpha/beta-inducible genes by double-stranded RNA. J. Interferon Res. 11, 131–141 (1991).

  132. 132.

    Daly, C. & Reich, N. C. Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon alpha/beta-stimulated genes. J. Biol. Chem. 270, 23739–23746 (1995).

  133. 133.

    Weaver, B. K., Kumar, K. P. & Reich, N. C. Interferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol. Cell. Biol. 18, 1359–1368 (1998).

  134. 134.

    Park, J. H. et al. Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification. Nat. Commun. 7, 12513 (2016).

  135. 135.

    Chiu, Y. H., Sun, Q. & Chen, Z. J. E1-L2 activates both ubiquitin and FAT10. Mol. Cell 27, 1014–1023 (2007).

  136. 136.

    Kim, K. I. et al. Ube1L and protein ISGylation are not essential for alpha/beta interferon signaling. Mol. Cell. Biol. 26, 472–479 (2006).

  137. 137.

    Krug, R. M., Zhao, C. & Beaudenon, S. Properties of the ISG15 E1 enzyme UbE1L. Methods Enzymol. 398, 32–40 (2005).

  138. 138.

    Zhao, C. et al. The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein. Proc. Natl Acad. Sci. USA 101, 7578–7582 (2004).

  139. 139.

    Kim, K. I., Giannakopoulos, N. V., Virgin, H. W. & Zhang, D. E. Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation. Mol. Cell. Biol. 24, 9592–9600 (2004).

  140. 140.

    Zou, W. & Zhang, D. E. The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase. J. Biol. Chem. 281, 3989–3994 (2006).

  141. 141.

    Okumura, F., Zou, W. & Zhang, D. E. ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP. Genes Dev. 21, 255–260 (2007).

  142. 142.

    Dastur, A., Beaudenon, S., Kelley, M., Krug, R. M. & Huibregtse, J. M. Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells. J. Biol. Chem. 281, 4334–4338 (2006).

  143. 143.

    Wong, J. J., Pung, Y. F., Sze, N. S. & Chin, K. C. HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets. Proc. Natl Acad. Sci. USA 103, 10735–10740 (2006).

  144. 144.

    Ketscher, L., Basters, A., Prinz, M. & Knobeloch, K. P. mHERC6 is the essential ISG15 E3 ligase in the murine system. Biochem. Biophys. Res. Commun. 417, 135–140 (2012).

  145. 145.

    Oudshoorn, D. et al. HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells. PLoS ONE 7, e29870 (2012).

  146. 146.

    Hare, N. J. et al. Microparticles released from Mycobacterium tuberculosis-infected human macrophages contain increased levels of the type I interferon inducible proteins including ISG15. Proteomics 15, 3020–3029 (2015).

  147. 147.

    Recht, M., Borden, E. C. & Knight, E. Jr. A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma. J. Immunol. 147, 2617–2623 (1991).

  148. 148.

    Zaheer, R. S. et al. Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity. Mucosal Immunol. 7, 1127–1138 (2014).

  149. 149.

    Arimoto, K. I. et al. STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling. Nat. Struct. Mol. Biol. 24, 279–289 (2017).

  150. 150.

    Rodriguez, M. R., Monte, K., Thackray, L. B. & Lenschow, D. J. ISG15 functions as an interferon-mediated antiviral effector early in the murine norovirus life cycle. J. Virol. 88, 9277–9286 (2014).

  151. 151.

    Dai, J., Pan, W. & Wang, P. ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro. Virol. J. 8, 468 (2011).

  152. 152.

    Kim, M. J. & Yoo, J. Y. Inhibition of hepatitis C virus replication by IFN-mediated ISGylation of HCV-NS5A. J. Immunol. 185, 4311–4318 (2010).

  153. 153.

    Tian, J. et al. Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes. Front. Microbiol. 6, 886 (2015).

  154. 154.

    Gonzalez-Sanz, R. et al. ISG15 is upregulated in respiratory syncytial virus infection and reduces virus growth through protein ISGylation. J. Virol. 90, 3428–3438 (2016).

  155. 155.

    Singh, P. K. et al. Zika virus infects cells lining the blood-retinal barrier and causes chorioretinal atrophy in mouse eyes. JCI Insight 2, e92340 (2017).

  156. 156.

    Hishiki, T. et al. Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication. Biochem. Biophys. Res. Commun. 448, 95–100 (2014).

  157. 157.

    Li, Y. et al. Interferon-stimulated gene 15 conjugation stimulates hepatitis B virus production independent of type I interferon signaling pathway in vitro. Mediators Inflamm. 2016, 7417648 (2016).

  158. 158.

    Chavoshi, S. et al. Identification of Kaposi sarcoma herpesvirus (KSHV) vIRF1 protein as a novel interaction partner of human deubiquitinase USP7. J. Biol. Chem. 291, 6281–6291 (2016).

  159. 159.

    Bianco, C. & Mohr, I. Restriction of HCMV replication by ISG15, a host effector regulated by cGAS-STING dsDNA sensing. J. Virol. 91, e02483–16 (2017).

  160. 160.

    Foy, E. et al. Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc. Natl Acad. Sci. USA 102, 2986–2991 (2005).

  161. 161.

    Sumpter, R. Jr. et al. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase. RIG-I. J. Virol. 79, 2689–2699 (2005).

  162. 162.

    Kim, M. J., Hwang, S. Y., Imaizumi, T. & Yoo, J. Y. Negative feedback regulation of RIG-I-mediated antiviral signaling by interferon-induced ISG15 conjugation. J. Virol. 82, 1474–1483 (2008).

  163. 163.

    Huang, Y. F., Wee, S., Gunaratne, J., Lane, D. P. & Bulavin, D. V. Isg15 controls p53 stability and functions. Cell Cycle 13, 2200–2210 (2014).

  164. 164.

    Park, J. M. et al. Modification of PCNA by ISG15 plays a crucial role in termination of error-prone translesion DNA synthesis. Mol. Cell 54, 626–638 (2014).

  165. 165.

    Lee, J. H. et al. Glycoprotein 90K, downregulated in advanced colorectal cancer tissues, interacts with CD9/CD82 and suppresses the Wnt/beta-catenin signal via ISGylation of beta-catenin. Gut 59, 907–917 (2010).

  166. 166.

    Yeh, Y. H., Yang, Y. C., Hsieh, M. Y., Yeh, Y. C. & Li, T. K. A negative feedback of the HIF-1alpha pathway via interferon-stimulated gene 15 and ISGylation. Clin. Cancer Res. 19, 5927–5939 (2013).

  167. 167.

    Im, E., Yoo, L., Hyun, M., Shin, W. H. & Chung, K. C. Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin. Open Biol. 6, 160193 (2016).

  168. 168.

    Cerikan, B. et al. Cell-intrinsic adaptation arising from chronic ablation of a key Rho GTPase regulator. Dev. Cell 39, 28–43 (2016).

  169. 169.

    Takeuchi, T. & Yokosawa, H. ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity. Biochem. Biophys. Res. Commun. 336, 9–13 (2005).

  170. 170.

    Takeuchi, T., Iwahara, S., Saeki, Y., Sasajima, H. & Yokosawa, H. Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme. J. Biochem. 138, 711–719 (2005).

  171. 171.

    Takeuchi, T., Kobayashi, T., Tamura, S. & Yokosawa, H. Negative regulation of protein phosphatase 2Cbeta by ISG15 conjugation. FEBS Lett. 580, 4521–4526 (2006).

  172. 172.

    Zou, W., Wang, J. & Zhang, D. E. Negative regulation of ISG15 E3 ligase EFP through its autoISGylation. Biochem. Biophys. Res. Commun. 354, 321–327 (2007).

  173. 173.

    Feng, Q. et al. UBE1L causes lung cancer growth suppression by targeting cyclin D1. Mol. Cancer Ther. 7, 3780–3788 (2008).

  174. 174.

    Shah, S. J. et al. UBE1L represses PML/RARα by targeting the PML domain for ISG15ylation. Mol. Cancer Ther. 7, 905–914 (2008).

  175. 175.

    Jeon, Y. J. et al. Chemosensitivity is controlled by p63 modification with ubiquitin-like protein ISG15. J. Clin. Invest. 122, 2622–2636 (2012).

Download references


The authors thank the members of the Lenschow laboratory for their critical reading of the manuscript during its preparation. The authors gratefully acknowledge support from the US National Institutes of Health (NIH) R01 AI080672 and Pew Charitable Trusts. Y.P. is funded through a Children Discovery Institute postdoctoral fellowship and the NIH postdoctoral training grant T32 CA009547.

Author information


  1. Department of Internal Medicine, Washington University School of Medicine, St Louis, MO, USA

    • Yi-Chieh Perng
    •  & Deborah J. Lenschow
  2. Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA

    • Deborah J. Lenschow


  1. Search for Yi-Chieh Perng in:

  2. Search for Deborah J. Lenschow in:


Y.P. researched data for the article. Y.P. and D.J.L. substantially contributed to discussion of content, wrote the article and reviewed and edited the manuscript before submission.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Deborah J. Lenschow.

Glossary terms


A small regulatory protein that can be added to a substrate protein by a process known as ubiquitylation and can alter the function of the substrate protein through degradation, localization and protein–protein interactions.

Ubiquitin-like modifiers

(Ubls). Small regulatory proteins that possess ubiquitin folds and are often conjugated onto a target protein similar to ubiquitin to alter function.

Genotoxic stressors

Agents that damage the genetic information within a cell, causing mutations or diseases.

Proteasome-mediated degradation

A cellular process to regulate the concentration of proteins and to degrade misfolded proteins by proteolysis, a chemical reaction that breaks peptide bonds.

Leader signal

A short peptide present at the amino terminus of newly synthesized proteins that are destined for the secretory pathway.

Secretory lysosomes

Dual-function organelles that could be used as a lysosome for degradation and hydrolysis and for storage of secretory proteins within the cell.


A double-stranded RNA helicase enzyme that functions as a cytosolic pattern-recognition receptor that recognizes short double-stranded or single-stranded RNA from viruses and triggers an antiviral response.

Usp18-knock-in mice

Mice in which the endogenous USP18 gene was replaced with a USP18 gene mutated so that it maintains its ability to bind to and inhibit signalling through the type I interferon receptor but its de-ISGylating capacity is lost, resulting in the accumulation of ISG15 conjugates.

Protein kinase R

(PKR). An interferon-induced, dsRNA-activated protein kinase that phosphorylates the eukaryotic translation initiation factor (eIF2α) in response to dsRNA and cellular stress, including viral infections.

Ovarian tumour domain

(OTU domain). A domain that is a shared protein region of a family of deubiquitylating proteolytic enzymes involved in processing of ubiquitin precursors.

Exosome secretion

A cellular secretion pathway mediated by the release of small membrane vesicles from multivesicular endosomes.

Viral latency

A type of persistent viral infection in which the pathogenic virus lies dormant without killing infected cells until it is reactivated by certain stimuli.

Aicardi–Goutières syndrome

(AGS). A rare, early-onset childhood inflammatory disorder characterized by elevated levels of type I interferons that results in skin and central nervous system manifestations.

Pathogen burden

The number of pathogens in an infected host that require the immune system for eradication.


An antiviral medication used to treat hepatitis C, respiratory syncytial virus and other viral infections.


A type of autophagy in which a defective and/or dysfunctional mitochondrion is selectively degraded by the lysosome.

Interferon-sensitive response element

(IRSE). A specific nucleotide sequence located in the promoters of interferon-stimulated genes (ISGs) that can bind to interferon stimulated gene factor 3 (ISGF3) or other transcriptional complexes upon type I interferon stimulation to initiation transcription of ISGs.

About this article

Publication history