Diversity of Ubiquitin and ISG15 Specificity among Nairoviruses' Viral Ovarian Tumor Domain Proteases

Abstract

Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferonstimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub-and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.

Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

“…Similar to cellular DUBs capable of forming a negative feedback system for the IFN responses (Kayagaki et al, 2007), virus-encoded DUBs of the ovarian tumor (OTU) superfamily are proposed to suppress the IFN responses. Viral OTU DUBs have been reported in both positive-strand ( Tymoviridae and Arteriviridae ) and negative-strand RNA viruses ( Bunyaviridae and tenuiviruses) (Capodagli et al, 2013; Frias-Staheli et al, 2007; Honig et al, 2004; van Kasteren et al, 2012; Kinsella et al, 2004; Lombardi et al, 2013; Makarova, 2000; Zhang et al, 2007). In addition to their deubiquitinase activity, certain viral DUBs can also remove interferon-stimulated gene 15 (ISG15) modifications, ubiquitin-like and interferon-inducible proteins that are conjugated to target proteins in a manner similar to Ub.…”

“…Other nairovirus OTUs appear to preferentially cleave either Ub or ISG15 moieties. For example, Dugbe virus displays a strong preference for Ub, whereas Erve virus displays a much higher activity towards ISG15 substrates (Capodagli et al, 2013). In contrast, the CCHFV OTU is capable of cleaving both Ub and ISG15 efficiently, and overexpression of the CCHFV OTU domain results in a general reduction of ubiquitinated and ISGylated protein levels.…”

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].…”

“…It takes a role in invasion of cells through antagonizing NF-jB signaling [1,20]. Viral OTU differs from mammalian counterparts by its broad deubiquitination and removal of ISG15 modification in the infected cells [1,3,5,7,9,10]. The crystal structure of viral OTU protease has been resolved along with ubiquitin (UB) and ISG15 proteins providing a structural approach to determine a pharmaceutical target site [2, 4-6, 8, 21, 22].…”

Identification of small molecule binding pocket for inhibition of Crimean–Congo hemorrhagic fever virus OTU protease

“…CCHFV OTU protease takes an important role in viral invasion through antagonizing NFkB signaling (Frias-Staheli et al, 2007;van Kasteren et al, 2012). The inhibition of antiviral activity by CCHFV OTU occurs by the removal of ubiquitin (UB) and interferonstimulated gene products 15 (ISG15), modifications occurring in a broad manner in the infected cells in contrast to target-specific OTU containing mammalian proteases (Frias-Staheli et al, 2007;Malakhova and Zhang, 2008;Akutsu et al, 2011;Capodagli et al, 2011Capodagli et al, , 2013. UB and ISG15 share a conserved C terminus motif 'LRLRGG' that is recognized by CCHFV OTU protease (Akutsu et al, 2011;Capodagli et al, 2011).…”