Molecular basis for ubiquitin and ISG15 cross-reactivity in viral ovarian tumor domains

Abstract

Crimean Congo hemorrhagic fever virus (CCHFV) is a deadly human pathogen that evades innate immune responses by efficiently interfering with antiviral signaling pathways mediated by NF-κB, IRF3, and IFNα/β. These pathways rely on protein ubiquitination for their activation, and one outcome is the modification of proteins with the ubiquitin (Ub)-like modifier interferon-stimulated gene (ISG)15. CCHFV and related viruses encode a deubiquitinase (DUB) of the ovarian tumor (OTU) family, which unlike eukaryotic OTU DUBs also targets ISG15 modifications. Here we characterized the viral OTU domain of CCHFV (vOTU) biochemically and structurally, revealing that it hydrolyzes four out of six tested Ub linkages, but lacks activity against linear and K29-linked Ub chains. vOTU cleaved Ub and ISG15 with similar kinetics, and we were able to understand vOTU cross-reactivity at the molecular level from crystal structures of vOTU in complex with Ub and ISG15. An N-terminal extension in vOTU not present in eukaryotic OTU binds to the hydrophobic Ile44 patch of Ub, which results in a dramatically different Ub orientation compared to a eukaryotic OTU-Ub complex. The C-terminal Ub-like fold of ISG15 (ISG15-C) adopts an equivalent binding orientation. Interestingly, ISG15-C contains an additional second hydrophobic surface that is specifically contacted by vOTU. These subtle differences in Ub/ISG15 binding allowed the design of vOTU variants specific for either Ub or ISG15, which will be useful tools to understand the relative contribution of ubiquitination vs. ISGylation in viral infection. Furthermore, the crystal structures will allow structure-based design of antiviral agents targeting this enzyme.cell signaling | structural biology | biochemistry | emerging disease

Crystal Structure of Human ISG15 Protein in Complex with Influenza B Virus NS1B

“…The ovarian tumor (OTU) domain proteases encoded by nairoviruses and arteriviruses help counteract both ubiquitylation and ISGylation by cleaving ubiquitin and ISG15 from target proteins (24). Structural studies showed that the ovarian tumor protease binds to the ubiquitin and C-terminal UBL domain of ISG15 (25,26). Previous biochemical study and the ISG15-NS1B complex structure shown here reveal that NS1B just targets the N-terminal UBL domain and the linker of ISG15 (9).…”

Ovine Fetal Immune Response to Cache Valley Virus Infection

“…In recent studies, ISG15 has been shown to conjugate to proteins in a manner similar to ubiquitin (40,41). Modifications and interference of antiviral signaling pathways involving ISG15 are mechanisms used by Crimean-Congo hemorrhagic fever virus, a bunyavirus, to evade the innate immune response (40). Upregulation of numerous ISG genes, including ISG15, and continuous stimulation of the innate antiviral response have been demonstrated in blood and tissues of bovine fetuses and cattle infected with BVDV (28,39).…”

“…Similar to the Mx protein, ISG15 is induced by type I IFN and has been associated with antiviral activity (29). In recent studies, ISG15 has been shown to conjugate to proteins in a manner similar to ubiquitin (40,41). Modifications and interference of antiviral signaling pathways involving ISG15 are mechanisms used by Crimean-Congo hemorrhagic fever virus, a bunyavirus, to evade the innate immune response (40).…”

Fluorometric CCHFV OTU protease assay with potent inhibitors

“…The tick-borne properties of the nairoviruses, and the large L segment existing in their genome make them different from other members of the Bunyaviridae viruses [1,2]. Crimean-Congo Hemorrhagic Fever (CCHF) constitutes a public health treat due to its high mortality rate in hospitalized patients [1][2][3][4][5][6][7][8][9][10]. It has been first reported in both the Crimea and Congo, since then there have been outbreaks in different parts of the world including Africa, Asia, and Eastern Europe [5,9,[11][12][13][14].…”