Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

Abstract

SignificanceAn understanding of the mechanisms by which viruses evade host immunity is essential to the development of antiviral drugs and viral detection strategies. Ubiquitin and ubiquitin-like modifications are crucial in cellular innate immune and infection responses and are often suppressed by viral proteins. We here identify a previously unknown mechanism of viral evasion. A viral protease, Lbpro, removes ubiquitin and the ubiquitin-like protein ISG15 incompletely from proteins. While this strategy efficiently and irreversibly shuts down these modification systems, it enables repurposing of tools and technologies developed for ubiquitin research in virus detection. Specifically, we show that foot-and-mouth disease virus infection can be detected using an anti-GlyGly antibody developed for ubiquitin mass spectrometry research.

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

“…1b). Viruses that encode viral proteases able to reverse ISG15 conjugation include nairoviruses [e.g., Crimean-Congo hemorrhagic fever virus (CCHFV), Nairobi sheep disease virus, and Erve virus) [39], arteriviruses [e.g., equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus] [39], picornaviruses (e.g., foot and mouth disease virus) [40], and coronaviruses [e.g., severe acute respiratory syndrome-related coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and mouse hepatitis virus] [41]. These cysteine proteases typically also possess substantial activity in reversing Ub conjugation and thus are often referred to as viral deubiquitinating proteins (DUBs).…”

“…In addition to the more extensively studied nairovirus OTUs and coronavirus PLPs, deISGylating activity has also been observed in arterivirus OTU-like PLPs, EAV PLP2, and the porcine reproductive and respiratory syndrome virus PLP2 [42,48e52]. Picornavirus Lb pro 's also possess deISGylating activity, and for foot and mouth disease virus, this is the most prominent deconjugating activity [40]. Overall, the widespread occurrence of viral proteins targeting ISG15 conjugation suggests that the ISG15evirus interface is a major factor impacting the balance between host responses and viral countermeasures.…”

Evaluating enzyme activities and structures of DUBs

“…For example, the Met1-specific DUB OTULIN requires substrate-assisted activation from a second ("proximal") Ub moiety, and thus will not react with a monoUb-based ABP (Keusekotten et al, 2013). In another unique case, the foot and mouth disease viral protease Lbpro hydrolyzes the UBL modifier ISG15 two residues short of the carboxy-terminus; conventional ISG15 ABPs place the electrophilic warhead out of register and will not react (Swatek et al, 2018). Although these are special cases of unique DUB mechanisms, they demonstrate the potential for false negative results in this assay.…”