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Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host's ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2'-5' oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shepard, Freitas, Rodriguez, Scholte, Baker, Hutchison, Longo, Miller, O’Boyle, Tandon, Zhao, Grimsey, Wells, Bergeron and Pegan.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, is a novel human betacoronavirus that is rapidly spreading worldwide. The outbreak currently includes over 3.7 million cases and 260,000 fatalities. As a betacoronavirus, SARS-CoV-2 encodes for a papain-like protease (PLpro) that is likely responsible for cleavage of the coronavirus (CoV) viral polypeptide. The PLpro is also responsible for suppression of host innate immune responses by virtue of its ability to reverse host ubiquitination and ISGylation events. Here, the biochemical activity of SARS-CoV-2 PLpro against ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) substrates is evaluated, revealing that the protease has a marked reduction in its ability to process K48 linked Ub substrates compared to its counterpart in SARS-CoV. Additionally, its substrate activity more closely mirrors that of the PLpro from the Middle East respiratory syndrome coronavirus and prefers ISG15s from certain species including humans. Additionally, naphthalene based PLpro inhibitors are shown to be effective at halting SARS-CoV-2 PLpro activity as well as SARS-CoV-2 replication.