Severe Acute and Middle East Respiratory syndrome coronaviruses (SARS-CoV and MERS-CoV) encode multifunctional papain-like proteases (PLPs) that have the ability to process the viral polyprotein to facilitate RNA replication as well as antagonize the host innate-immune response. The latter function involves reversing post-translational modification of cellular proteins conjugated with either ubiquitin (Ub) or Ub-like interferon stimulated gene product 15 (ISG15). Ubiquitin is known to be highly conserved among eukaryotes but surprisingly ISG15 is highly divergent among animals. The ramifications of this sequence divergence to recognition of ISG15 by coronaviral papain-like protease at the structural and biochemical levels are poorly understood. Therefore, the activity of PLPs from SARS-CoV, MERS-CoV and mouse hepatitis virus (MHV) was evaluated against seven ISG15s originating from an assortment of animal species susceptible, and not, to certain coronavirus infections. Excitingly, our kinetic, thermodynamic and structural analysis revealed an array of different preferences among PLPs. Included in these studies is the first insight into a coronoavirus PLP’s interface with ISG15 via SARS-CoV PLP in complex with the principle binding domain of human and mouse ISG15s. The first X-ray structure of the full-length mouse ISG15 protein is also reported and highlights a unique, twisted-hinge region of ISG15 that is not conserved in human ISG15 suggesting a potential role in differential recognition. Taken together, this new information provides a structural and biochemical understanding of the distinct specificities amongst coronavirus PLPs observed and addresses a critical gap of how PLPs can interact with ISG15s from a wide variety of species.

Interferon-stimulated gene product 15 (ISG15) is a key component of host responses to microbial infection. Despite having been known for four decades, grasping the functions and features of ISG15 has been a slow and elusive process. Substantial work over the past two decades has greatly enhanced this understanding, revealing the complex and variable nature of this protein. This has unveiled multiple mechanisms of action that are only now beginning to be understood. In addition, it has uncovered diversity not only between how ISG15 affects different pathogens but also between the function and structure of ISG15 itself between different host species. Here we review the complexity of ISG15 within the context of viral infection, focusing primarily on its antiviral function and the mechanisms viruses employ to thwart its effects. We highlight what is known regarding the impact of ISG15 sequence and structural diversity on these interactions and discuss the aspects presenting the next frontier toward elucidating a more complete picture of ISG15 function.

SUMMARY Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and supports the development of therapeutics and vaccines.

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.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 Å in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs. Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.