PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses.

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses.

Design, synthesis and evaluation of inhibitors of the SARS-CoV-2 nsp3 macrodomain.

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening.

The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100 µM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways­interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)­activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung­derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV2 nsp3 Macrodomain.

A series of amino acid based 7H -pyrrolo[2,3- d ]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp 22 and the amide backbone NH of Ile 23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe 157 and Asp 156 , part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Unique Mutations in the Murine Hepatitis Virus Macrodomain Differentially Attenuate Virus Replication, Indicating Multiple Roles for the Macrodomain in Coronavirus Replication.

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in nonstructural protein 3 (nsp3) that binds and hydrolyzes mono-ADP-ribose (MAR) covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutation of a highly conserved asparagine residue, which makes contact with the distal ribose of ADP-ribose. To determine if additional Mac1 activities contribute to CoV replication, we compared the replication of murine hepatitis virus (MHV) Mac1 mutants, D1329A and N1465A, to the previously mentioned asparagine mutant, N1347A. These residues contact the adenine and proximal ribose in ADP-ribose, respectively. N1465A had no effect on MHV replication or pathogenesis, while D1329A and N1347A both replicated poorly in bone marrow-derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. Interestingly, D1329A was also significantly more attenuated than N1347A in all cell lines tested. Conversely, D1329A retained some ability to block beta interferon (IFN-ß) transcript accumulation compared to N1347A, indicating that these mutations have different effects on Mac1 functions. Combining these two mutations resulted in a virus that was unrecoverable, suggesting that the combined activities of Mac1 are essential for MHV replication. We conclude that Mac1 has multiple functions that promote the replication of MHV, and that these results provide further evidence that Mac1 is a prominent target for anti-CoV therapeutics. IMPORTANCE In the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within nonstructural protein 3. It has received significant attention as a potential drug target, as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the functions of Mac1 during infection remain largely unknown. Here, using targeted mutations in different regions of Mac1, we found that Mac1 has multiple functions that promote the replication of MHV, a model CoV, and, therefore, is more important for MHV replication than previously appreciated. These results will help guide the discovery of these novel functions of Mac1 and the development of inhibitory compounds targeting this domain.

The SARS-CoV-2 Conserved Macrodomain Is a Mono-ADP-Ribosylhydrolase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity.

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+ Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

The Viral Macrodomain Counters Host Antiviral ADP-Ribosylation.

Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune responses to virus infection. ADP-ribose is covalently attached to target proteins by poly-ADP-ribose polymerases (PARPs), using nicotinamide adenine dinucleotide (NAD+) as a substrate. This modification can have a wide variety of effects on proteins including alteration of enzyme activity, protein-protein interactions, and protein stability. Several PARPs are induced by interferon (IFN) and are known to have antiviral properties, implicating ADP-ribosylation in the host defense response and suggesting that viral macrodomains may counter this response. Recent studies have demonstrated that viral macrodomains do counter the innate immune response by interfering with PARP-mediated antiviral defenses, stress granule formation, and pro-inflammatory cytokine production. Here, we will describe the known functions of the viral macrodomains and review recent literature demonstrating their roles in countering PARP-mediated antiviral responses.

The impact of PARPs and ADP-ribosylation on inflammation and host-pathogen interactions.

Poly-adenosine diphosphate-ribose polymerases (PARPs) promote ADP-ribosylation, a highly conserved, fundamental posttranslational modification (PTM). PARP catalytic domains transfer the ADP-ribose moiety from NAD+ to amino acid residues of target proteins, leading to mono- or poly-ADP-ribosylation (MARylation or PARylation). This PTM regulates various key biological and pathological processes. In this review, we focus on the roles of the PARP family members in inflammation and host-pathogen interactions. Here we give an overview the current understanding of the mechanisms by which PARPs promote or suppress proinflammatory activation of macrophages, and various roles PARPs play in virus infections. We also demonstrate how innovative technologies, such as proteomics and systems biology, help to advance this research field and describe unanswered questions.

Murine Coronavirus Infection Activates the Aryl Hydrocarbon Receptor in an Indoleamine 2,3-Dioxygenase-Independent Manner, Contributing to Cytokine Modulation and Proviral TCDD-Inducible-PARP Expression.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1ß (IL-1ß), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1-/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis.IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

IFN-I response timing relative to virus replication determines MERS coronavirus infection outcomes.

Type 1 IFNs (IFN-I) generally protect mammalian hosts from virus infections, but in some cases, IFN-I is pathogenic. Because IFN-I is protective, it is commonly used to treat virus infections for which no specific approved drug or vaccine is available. The Middle East respiratory syndrome-coronavirus (MERS-CoV) is such an infection, yet little is known about the role of IFN-I in this setting. Here, we show that IFN-I signaling is protective during MERS-CoV infection. Blocking IFN-I signaling resulted in delayed virus clearance, enhanced neutrophil infiltration, and impaired MERS-CoV-specific T cell responses. Notably, IFN-I administration within 1 day after infection (before virus titers peak) protected mice from lethal infection, despite a decrease in IFN-stimulated gene (ISG) and inflammatory cytokine gene expression. In contrast, delayed IFN-ß treatment failed to effectively inhibit virus replication, increased infiltration and activation of monocytes, macrophages, and neutrophils in the lungs, and enhanced proinflammatory cytokine expression, resulting in fatal pneumonia in an otherwise sublethal infection. Together, these results suggest that the relative timing of the IFN-I response and maximal virus replication is key in determining outcomes, at least in infected mice. By extension, IFN-αß or combination therapy may need to be used cautiously to treat viral infections in clinical settings.

Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element.

There are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections. MERS-CoV 3CL protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting MERS-CoV. We describe herein the structure-guided design and evaluation of a novel class of inhibitors of MERS-CoV 3CL protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and PK properties. The mechanism of action of the compounds and the structural determinants associated with binding were illuminated using X-ray crystallography.

Viral Macrodomains: Unique Mediators of Viral Replication and Pathogenesis.

Viruses from the Coronaviridae, Togaviridae, and Hepeviridae families ​all contain genes that encode a conserved protein domain, called a macrodomain; however, the role of this domain during infection has remained enigmatic. The recent discovery that mammalian macrodomain proteins enzymatically remove ADP-ribose, a common post-translation modification, from proteins has led to an outburst of studies describing both the enzymatic activity and function of viral macrodomains. These new studies have defined these domains as de-ADP-ribosylating enzymes, which indicates that these viruses have evolved to counteract antiviral ADP-ribosylation, likely mediated by poly-ADP-ribose polymerases (PARPs). Here, we comprehensively review this rapidly expanding field, describing the structures and enzymatic activities of viral macrodomains, and discussing their roles in viral replication and pathogenesis.

Virus-induced inflammasome activation is suppressed by prostaglandin D2/DP1 signaling.

Prostaglandin D2 (PGD2), an eicosanoid with both pro- and anti-inflammatory properties, is the most abundantly expressed prostaglandin in the brain. Here we show that PGD2 signaling through the D-prostanoid receptor 1 (DP1) receptor is necessary for optimal microglia/macrophage activation and IFN expression after infection with a neurotropic coronavirus. Genome-wide expression analyses indicated that PGD2/DP1 signaling is required for up-regulation of a putative inflammasome inhibitor, PYDC3, in CD11b+ cells in the CNS of infected mice. Our results also demonstrated that, in addition to PGD2/DP1 signaling, type 1 IFN (IFN-I) signaling is required for PYDC3 expression. In the absence of Pydc3 up-regulation, IL-1ß expression and, subsequently, mortality were increased in infected DP1-/- mice. Notably, survival was enhanced by IL1 receptor blockade, indicating that the effects of the absence of DP1 signaling on clinical outcomes were mediated, at least in part, by inflammasomes. Using bone marrow-derived macrophages in vitro, we confirmed that PYDC3 expression is dependent upon DP1 signaling and that IFN priming is critical for PYDC3 up-regulation. In addition, Pydc3 silencing or overexpression augmented or diminished IL-1ß secretion, respectively. Furthermore, DP1 signaling in human macrophages also resulted in the up-regulation of a putative functional analog, POP3, suggesting that PGD2 similarly modulates inflammasomes in human cells. These findings demonstrate a previously undescribed role for prostaglandin signaling in preventing excessive inflammasome activation and, together with previously published results, suggest that eicosanoids and inflammasomes are reciprocally regulated.

The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection.

ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3's de-ADP-ribosylation activity in vitro Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs. IMPORTANCE: The macrodomain is a ubiquitous structural domain that removes ADP-ribose from proteins, reversing the activity of ADP-ribosyltransferases. All coronaviruses contain a macrodomain, suggesting that ADP-ribosylation impacts coronavirus infection. However, its function during infection remains unknown. Here, we found that the macrodomain is an important virulence factor for a highly pathogenic human CoV, SARS-CoV. Viruses with macrodomain mutations that abrogate its ability to remove ADP-ribose from protein were unable to cause lethal disease in mice. Importantly, the SARS-CoV macrodomain suppressed the innate immune response during infection. Our data suggest that an early innate immune response can protect mice from lethal disease. Understanding the mechanism used by this enzyme to promote disease will open up novel avenues for coronavirus therapies and give further insight into the role of macrodomains in viral pathogenesis.

Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice.

Highly pathogenic human respiratory coronaviruses cause acute lethal disease characterized by exuberant inflammatory responses and lung damage. However, the factors leading to lung pathology are not well understood. Using mice infected with SARS (severe acute respiratory syndrome)-CoV, we show that robust virus replication accompanied by delayed type I interferon (IFN-I) signaling orchestrates inflammatory responses and lung immunopathology with diminished survival. IFN-I remains detectable until after virus titers peak, but early IFN-I administration ameliorates immunopathology. This delayed IFN-I signaling promotes the accumulation of pathogenic inflammatory monocyte-macrophages (IMMs), resulting in elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific T cell responses. Genetic ablation of the IFN-αß receptor (IFNAR) or IMM depletion protects mice from lethal infection, without affecting viral load. These results demonstrate that IFN-I and IMM promote lethal SARS-CoV infection and identify IFN-I and IMMs as potential therapeutic targets in patients infected with pathogenic coronavirus and perhaps other respiratory viruses.

The nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis.

UNLABELLED: All coronaviruses encode a macrodomain containing ADP-ribose-1"-phosphatase (ADRP) activity within the N terminus of nonstructural protein 3 (nsp3). Previous work showed that mouse hepatitis virus strain A59 (MHV-A59) with a mutated catalytic site (N1348A) replicated similarly to wild-type virus but was unable to cause acute hepatitis in mice. To determine whether this attenuated phenotype is applicable to multiple disease models, we mutated the catalytic residue in the JHM strain of MHV (JHMV), which causes acute and chronic encephalomyelitis, using a newly developed bacterial artificial chromosome (BAC)-based MHV reverse genetics system. Infection of mice with the macrodomain catalytic point mutant virus (N1347A) resulted in reductions in lethality, weight loss, viral titers, proinflammatory cytokine and chemokine expression, and immune cell infiltration in the brain compared to mice infected with wild-type virus. Specifically, macrophages were most affected, with approximately 2.5-fold fewer macrophages at day 5 postinfection in N1347A-infected brains. Tumor necrosis factor (TNF) and interferon (IFN) signaling were not required for effective host control of mutant virus as all N1347A virus-infected mice survived the infection. However, the adaptive immune system was required for protection since N1347A virus was able to cause lethal encephalitis in RAG1(-/-) (recombination activation gene 1 knockout) mice although disease onset was modestly delayed. Overall, these results indicate that the BAC-based MHV reverse genetics system will be useful for studies of JHMV and expand upon previous studies, showing that the macrodomain is critical for the ability of coronaviruses to evade the immune system and promote viral pathogenesis. IMPORTANCE: Coronaviruses are an important cause of human and veterinary diseases worldwide. Viral processes that are conserved across a family are likely to be good targets for the development of antiviral therapeutics and vaccines. The macrodomain is a ubiquitous structural domain and is also conserved among all coronaviruses. The coronavirus macrodomain has ADP-ribose-1"-phosphatase activity; however, its function during infection remains unclear as does the reason that coronaviruses have maintained this enzymatic activity throughout evolution. For MHV, this domain has now been shown to promote multiple types of disease, including hepatitis and encephalitis. These data indicate that this domain is vital for the virus to replicate and cause disease. Understanding the mechanism used by this enzyme to promote viral pathogenesis will open up novel avenues for therapies and may give further insight into the role of macrodomain proteins in the host cell since these proteins are found in all living organisms.