The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Anthony R Fehr - One of the best experts on this subject based on the ideXlab platform.
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the viral Macrodomain counters host antiviral adp ribosylation
Viruses, 2020Co-Authors: Yousef M O Alhammad, Anthony R FehrAbstract: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.
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the coronavirus Macrodomain is required to prevent parp mediated inhibition of virus replication and enhancement of ifn expression
PLOS Pathogens, 2019Co-Authors: Matthew E Grunewald, Robert Lease, Christopher S. Sullivan, Yating Chen, Chad V. Kuny, Stanley Perlman, Takashi Maejima, Masanori Aikawa, Dana Ferraris, Anthony R FehrAbstract:ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for Macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that Macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with Macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the Macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production.
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Viral Macrodomains: Unique Mediators of Viral Replication and Pathogenesis
Trends in Microbiology, 2017Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Stanley PerlmanAbstract: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.
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The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection.
Mbio, 2016Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Rudragouda Channappanavar, Craig Fett, Jincun Zhao, Jeremiah Athmer, David K. Meyerholz, Stanley PerlmanAbstract:ABSTRACT 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.
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The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection
American Society for Microbiology, 2016Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Rudragouda Channappanavar, Craig Fett, Jincun Zhao, Jeremiah Athmer, David K. Meyerholz, Stanley PerlmanAbstract: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
Stanley Perlman - One of the best experts on this subject based on the ideXlab platform.
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the coronavirus Macrodomain is required to prevent parp mediated inhibition of virus replication and enhancement of ifn expression
PLOS Pathogens, 2019Co-Authors: Matthew E Grunewald, Robert Lease, Christopher S. Sullivan, Yating Chen, Chad V. Kuny, Stanley Perlman, Takashi Maejima, Masanori Aikawa, Dana Ferraris, Anthony R FehrAbstract:ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for Macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that Macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with Macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the Macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production.
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Viral Macrodomains: Unique Mediators of Viral Replication and Pathogenesis
Trends in Microbiology, 2017Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Stanley PerlmanAbstract: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.
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The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection.
Mbio, 2016Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Rudragouda Channappanavar, Craig Fett, Jincun Zhao, Jeremiah Athmer, David K. Meyerholz, Stanley PerlmanAbstract:ABSTRACT 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.
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The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection
American Society for Microbiology, 2016Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Rudragouda Channappanavar, Craig Fett, Jincun Zhao, Jeremiah Athmer, David K. Meyerholz, Stanley PerlmanAbstract: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
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the nsp3 Macrodomain promotes virulence in mice with coronavirus induced encephalitis
Journal of Virology, 2015Co-Authors: Anthony R Fehr, Rudragouda Channappanavar, Jeremiah Athmer, David K. Meyerholz, Judith M Phillips, Stanley PerlmanAbstract:ABSTRACT 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.
Andrew Harold Limper - One of the best experts on this subject based on the ideXlab platform.
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primary alveolar epithelial cell surface membrane microdomain function is required for pneumocystis β glucan induced inflammatory responses
Innate Immunity, 2012Co-Authors: Scott E Evans, Theodore J Kottom, Richard E. Pagano, Andrew Harold LimperAbstract:Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent b-(1!3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-a and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that antimicrodomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.
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primary alveolar epithelial cell surface membrane microdomain function is required for pneumocystis β glucan induced inflammatory responses
Innate Immunity, 2012Co-Authors: Scott E Evans, Theodore J Kottom, Richard E. Pagano, Andrew Harold LimperAbstract:Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent b-(1!3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-a and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that antimicrodomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.
Yousef M O Alhammad - One of the best experts on this subject based on the ideXlab platform.
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the sars cov 2 conserved Macrodomain is a mono adp ribosylhydrolase
Journal of Virology, 2021Co-Authors: Yousef M O Alhammad, Maithri M Kashipathy, Anuradha Roy, Jeanphilippe Gagne, Peter R Mcdonald, Philip Gao, Louis Nonfoux, Kevin P Battaile, David K Johnson, Erik D HolmstromAbstract:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem Macrodomains within non-structural 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 anti-viral 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 MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low µM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, 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 compared to 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 greater than 1.2 million deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved Macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly 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 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.
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the sars cov 2 conserved Macrodomain is a mono adp ribosylhydrolase
bioRxiv, 2020Co-Authors: Yousef M O Alhammad, Maithri M Kashipathy, Anuradha Roy, Jeanphilippe Gagne, Peter R Mcdonald, Philip Gao, Louis Nonfoux, Kevin P Battaile, David K Johnson, Erik D HolmstromAbstract:ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem Macrodomains within non-structural 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 anti-viral 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 MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, 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 compared to 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 greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved Macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly 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 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.
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the sars cov 2 conserved Macrodomain is a highly efficient adp ribosylhydrolase enzyme
bioRxiv, 2020Co-Authors: Yousef M O Alhammad, Maithri M Kashipathy, Anuradha Roy, Jeanphilippe Gagne, Peter R Mcdonald, Philip Gao, Louis Nonfoux, Kevin P Battaile, David K Johnson, Erik D HolmstromAbstract:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem Macrodomains within non-structural protein 3 (nsp3). The first of these Macrodomains, termed Mac1, is conserved throughout CoVs, binds to mono- and poly-ADP-ribose, and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating this domain as a prominent virulence factor and potential therapeutic target. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose refined at 2.2 A resolution. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and ADP-ribose binding modes as shown by structural comparison. All three CoV Mac1 proteins bound to ADP-ribose with low μM affinities. They also demonstrated highly efficient de-MARylating activity, which was greater than that of the human Mdo2 Macrodomain. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are highly efficient ADP-ribosylhydrolases with strikingly similar activity, indicating that compounds targeting CoV Mac1 proteins may have broad antiviral activity against CoVs.
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the viral Macrodomain counters host antiviral adp ribosylation
Viruses, 2020Co-Authors: Yousef M O Alhammad, Anthony R FehrAbstract: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.
S Knapp - One of the best experts on this subject based on the ideXlab platform.
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structural insights into plasticity and discovery of remdesivir metabolite gs 441524 binding in sars cov 2 Macrodomain
ACS Medicinal Chemistry Letters, 2021Co-Authors: Martin Schroder, Vincent Olieric, M Sharpe, Victor Hernandezolmos, Ewgenij Proschak, Daniel Merk, S KnappAbstract:The nsP3 Macrodomain is a conserved protein interaction module that plays essential regulatory roles in the host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus targeting this protein domain may offer a therapeutic strategy to combat current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of Macrodomain crystal structures complexed with diverse naturally occurring nucleotides, small molecules, and nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 Macrodomain structural plasticity and provide chemical starting points for future inhibitor development.
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structural insights into plasticity and discovery of remdesivir metabolite gs 441524 binding in sars cov 2 Macrodomain
bioRxiv, 2021Co-Authors: Vincent Olieric, M Sharpe, Ewgenij Proschak, Daniel Merk, Martin Schroeder, Victor Olmos, S KnappAbstract:The nsP3 Macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of Macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 Macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.
