Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating liveattenuated vaccines.

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies.

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a non-covalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multi-component Ugi reaction was utilized to rapidly explore structure activity relationships within S1′, S1, and S2 enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a non-covalent mechanism of action.

Coronaviruses encode papain-like proteases (PLpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (DUB)/deISGylating activity, which is hypothesized to modify the innate immune response to infection. Here, we investigate the predicted DUB activity of the PLpro domain of the recently described Middle East Respiratory Syndrome Coronavirus (MERS-CoV). We found that expression of MERS-CoV PLpro reduces the levels of ubiquitinated and ISGylated host cell proteins; consistent with multifunctional PLpro activity. Further, we compared the ability of MERS-CoV PLpro and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) PLpro to block innate immune signaling of proinflammatory cytokines. We show that expression of SARS-CoV and MERS-CoV PLpros blocks upregulation of cytokines CCL5, IFN-β and CXCL10 in stimulated cells. Overall these results indicate that the PLpro domains of MERS-CoV and SARS-CoV have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis.

Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), came the realization of the multifunctional nature of these enzymes. Structural and enzymatic studies revealed that SARS-CoV PLpro can act as both a protease to cleave peptide bonds and also as a deubiquitinating (DUB) enzyme to cleave the isopeptide bonds found in polyubiquitin chains. Furthermore, viral DUBs can also remove the protective effect of conjugated ubiquitin-like molecules such as interferon stimulated gene 15 (ISG15). Extension of these studies to other coronaviruses and arteriviruses led to the realization that viral protease/DUB activity is conserved in many family members. Overexpression studies revealed that viral protease/DUB activity can modulate or block activation of the innate immune response pathway. Importantly, mutations that alter DUB activity but not viral protease activity have been identified and arteriviruses expressing DUB mutants stimulated higher levels of acute inflammatory cytokines after infection. Further understanding of the multifunctional nature of the Nidovirus PLP/DUBs may facilitate vaccine development. Here, we review studies describing the PLPs’ enzymatic activity and their role in virus pathogenesis.

The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48-and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48-and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition. IMPORTANCEUnlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates, but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anti-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated, as well as insight into the design of inhibitors against MERS-CoV PLpro.

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.

Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.