Biochemical and Structural Insights into the Preference of Nairoviral DeISGylases for Interferon-Stimulated Gene Product 15 Originating from Certain Species

Abstract

The regulation of the interferon type I (IFN-I) response has been shown to rely on posttranslational modification by ubiquitin (Ub) and Ub-like interferon-stimulated gene product 15 (ISG15) to stabilize, or activate, a variety of IFN-I signaling and downstream effector proteins. Unlike Ub, which is almost perfectly conserved among eukaryotes, ISG15 is highly divergent, even among mammals. Since zoonotic viruses rely on viral proteins to recognize, or cleave, ISG15 conjugates in order to evade, or suppress, innate immunity, the impact of ISG15 biodiversity on deISGylating proteases of the ovarian tumor family (vOTU) from nairoviruses was evaluated. The enzymatic activities of vOTUs originating from the Crimean-Congo hemorrhagic fever virus, Erve virus, and Nairobi sheep disease virus were tested against ISG15s from humans, mice, shrews, sheep, bats, and camels, which are mammalian species known to be infected by nairoviruses. This along with investigation of binding by isothermal titration calorimetry illustrated significant differences in the abilities of nairovirus deISGylases to accommodate certain species of ISG15. To investigate the molecular underpinnings of species preferences of these vOTUs, a structure was determined to 2.5 Å for a complex of Erve virus vOTU protease and a mouse ISG15 domain. This structure revealed the molecular basis of Erve virus vOTU's preference for ISG15 over Ub and the first structural insight into a nonhuman ISG15. This structure also revealed key interactions, or lack thereof, surrounding three amino acids that may drive a viral deISgylase to prefer an ISG15 from one species over that of another. IMPORTANCEViral ovarian tumor domain proteases (vOTUs) are one of the two principal classes of viral proteases observed to reverse posttranslational modification of host proteins by ubiquitin and interferon-stimulated gene product 15 (ISG15), subsequently facilitating downregulation of IFN-I signaling pathways. Unlike the case with ubiquitin, the amino acid sequences of ISG15s from various species are notably divergent. We illustrate that vOTUs have clear preferences for ISG15s from certain species. In addition, these observations are related to the molecular insights acquired via the first X-ray structure of the vOTU from the Erve nairovirus in complex with the first structurally resolved nonhuman ISG15. This information implicates certain amino acids that drive the preference of vOTUs for ISG15s from certain species.

Approaches for investigating the extracellular signaling function of ISG15

“…It should be noted that ISGylation does not target proteins for proteasomal degradation and poly-ISGylated chains do not appear to be formed in cells. The single de-ISGylating enzyme for ISG15 is human Usp18 (mouse Ubp43) (Malakhov, Malakhova, Kim, Ritchie, & Zhang, 2002), although several virally encoded de-ISGylating enzymes have been identified, consistent with the hypothesis that ISGylation is antiviral (Akutsu, Ye, Virdee, Chin, & Komander, 2011;Daczkowski, Goodwin, Dzimianski, Farhat, & Pegan, 2017;Deaton et al, 2016;Swatek et al, 2018). Importantly, all three conjugation enzymes (UBA7, Ube2L6, and Herc5) as well as Usp18 are induced at the transcriptional level by Type 1 IFN signaling.…”

“…All altered proteins were soluble when purified as described later and they were expressed at similar levels to wild-type ISG15. It should be noted that ISG15 has been crystallized with several interacting partners including Influenza B NS1 (NS1B), Usp18, and viral de-ISGylases (Akutsu et al, 2011;Basters et al, 2017;Deaton et al, 2016;Swatek et al, 2018;Zhao et al, 2016). Residues necessary for interaction with NS1B are located within the N-terminal ubiquitin-like domain of ISG15, while the residues for interactions with the de-ISGylases are located on the C-terminal lobe.…”

ISG15: It's Complicated

“…Although Influenza B via the NS1 protein is the best-characterized example of a virus affected by ISG15 interspecies diversity, recent studies indicate that the impact may extend to other viruses. Nairovirus OTUs have been observed to interact with ISG15s from different species to different degrees in vitro [77,78]. Comparison between crystal structures of the Erve virus OTU bound to the Cterminal Ubl domain of mouse ISG15 and the OTU of CCHFV bound to human ISG15 provided insights into the possible contributing factors to these differences.…”

Structure and Function of Viral Deubiquitinating Enzymes

“…In contrast to CCHFV and DUGV OTU domains, the ERVV OTU domain is essentially free of DUB activity, possessing only activity against ISG15, with a particularly high affinity for human ISG15 as determined by isothermal titration calorimetry [221,224]. Analysis of a crystal structure of ERVV OTU in complex with mISG15 attributed the inability of ERVV OTU to bind Ub to the presence of an electrophilic region (Arg21, Ser132, Asn134) at the interface that binds ISG15, which appears to be able to accommodate ISG15 residue Glu87, but not the spatially conserved hydrophobic Ub residue Leu8 [224].…”