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.

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5′-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wildtype−infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.C oronaviruses (CoVs) are positive-sense RNA viruses that replicate in the cytoplasm of infected cells. The positivesense virion RNA is translated to generate the viral replication machinery, which then replicates the positive-sense RNA into negative-sense, genomic RNA and subgenomic RNAs (sgRNAs). The negative-sense RNAs then function as templates for synthesis of positive-sense genomic RNA and sgRNA (1, 2). This replication strategy can generate long double-stranded RNA (dsRNA) intermediates (3), that may act as pathogen-associated molecular patterns (PAMPs) recognized by cytoplasmic pattern recognition receptors (PRRs) (4, 5). The specific PRR that recognizes CoV RNA is MDA5, which can activate the type I interferon (IFN) response in macrophages (6). Other host dsRNA PRRs, such as PKR and OAS, are also activated and operate to limit CoV replication (7-11). CoVs encode multiple proteins that antagonize these innate immune responses, particularly the activation of the IFN response (9, 12-16), ultimately leading to a dysregulated immune response and increased immunopathogenesis (17,18). Understanding the mechanisms used by CoVs to delay IFN signaling may provide opportunities for the development of antivirals and live-attenuated vaccines to limit CoV infections.Here, we investigate the mechanism used by one CoV IFN antagonist, the nonstructural protein 15 (nsp15), which is an endoribonuclease designated EndoU. EndoU is highly conserved in all known CoVs (19,20). EndoU is similar to the cellular endoribonuclease XendoU, as revealed by bioinformatic analysis of the amino acid sequence (21). X-ray structures of EndoU revealed conserved endoribonuclease folds with catalytic histidine residues required for endoribonuclease activity (22)(23)(24)(25)(26). Purified EndoU was shown to cleave single-str...

We have developed a Kramers-Kronig consistent analytical expression to fit the measured optical functions of hydrogenated amorphous silicon (a-Si:H) based alloys, i.e., the real and imaginary parts of the dielectric function (⑀ 1 ,⑀ 2) ͑or the index of refraction n and absorption coefficient ␣͒ versus photon energy E for the alloys. The alloys of interest include amorphous silicon-germanium (a-Si 1Ϫx Ge x :H) and silicon-carbon (a-Si 1Ϫx C x :H), with band gaps ranging continuously from ϳ1.30 to 1.95 eV. The analytical expression incorporates the minimum number of physically meaningful, E independent parameters required to fit (⑀ 1 ,⑀ 2) versus E. The fit is performed simultaneously throughout the following three regions: ͑i͒ the below-band gap ͑or Urbach tail͒ region where ␣ increases exponentially with E, ͑ii͒ the near-band gap region where transitions are assumed to occur between parabolic bands with constant dipole matrix element, and ͑iii͒ the above-band gap region where (⑀ 1 ,⑀ 2) can be simulated assuming a single Lorentz oscillator. The expression developed here provides an improved description of ⑀ 2 ͑or ␣͒ in the below-band gap and near-band gap regions compared with previous approaches. Although the expression is more complicated analytically, it has numerous applications in the analysis and simulation of thin film a-Si:H based p-in and n-i-p multilayer photovoltaic devices. First, we describe an approach whereby, from a single accessible measure of the optical band gap, the optical functions can be generated over the full solar spectrum for a sample set consisting of the highest quality intrinsic a-Si:H based alloys prepared by plasma-enhanced chemical vapor deposition using the principle of maximal H 2 dilution. Second, we describe quantitatively how such an approach can be modified for sample sets consisting of lower quality alloy materials. Finally, we demonstrate how the generated optical functions can be used in simulations of the absorption, reflection, and quantum efficiency spectra of a-Si:H based single-junction and multijunction solar cells.

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.

Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis are critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN)-deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt, or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea; however, there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing the IFN response in epithelial cells and macrophages, which facilitates replication, shedding, and pathogenesis in vivo. We conclude that PEDV En-doU activity is a key virulence factor that suppresses both type I and type III IFN responses. IMPORTANCE Coronaviruses (CoVs) can emerge from an animal reservoir into a naive host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus (alpha-CoV), infects gut epithelial cells and macrophages, inducing diarrhea and resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon response and the type III interferon response in macrophages and epithelial cells. This activation of interferon resulted in limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.