Mutagenesis ofS-Adenosyl-l-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity

ABSTRACTEukaryotic mRNAs possess a methylated 5′-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5′ caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conservedS-adenosyl-l-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activityin vitro. However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon (IFN), and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to wild-type (WT) MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with IFN-β, and nsp14 G332A genomes were translated less efficiently bothin vitroand during viral infection. In contrast, substitution of alanine at MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by IFN-β compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding-site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differin vitroand during virus infection.IMPORTANCEHuman coronaviruses, most notably severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that substitution of alanine at G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.

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Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis

Journal of Virology ◽

10.1128/jvi.02140-18 ◽

2019 ◽

Vol 93 (12) ◽

Cited By ~ 7

Author(s):

Xufang Deng ◽

Robert C. Mettelman ◽

Amornrat O’Brien ◽

John A. Thompson ◽

Timothy E. O’Brien ◽

...

Keyword(s):

Viral Replication ◽

Reverse Genetics ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Temperature Sensitive ◽

Type I ◽

Polyubiquitin Chains ◽

Host Innate Immune Response ◽

Necessary And Sufficient

ABSTRACTAnalysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferonin vitroand are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCECoronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

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Mutations in Coronavirus Nonstructural Protein 10 Decrease Virus Replication Fidelity

Journal of Virology ◽

10.1128/jvi.00110-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6418-6426 ◽

Cited By ~ 27

Author(s):

Everett Clinton Smith ◽

James Brett Case ◽

Hervé Blanc ◽

Ofer Isakov ◽

Noam Shomron ◽

...

Keyword(s):

Virus Replication ◽

Viral Protein ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Nucleoside Analogs ◽

Murine Hepatitis Virus ◽

Replication Fidelity ◽

Peak Titer ◽

Substitution Mutations

ABSTRACTCoronaviruses (CoVs) are unique in encoding a 3′→5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication, likely via proofreading. nsp14 associates with the CoV RNA-dependent RNA polymerase (nsp12-RdRp), and nsp14-ExoN activity is enhanced by binding nsp10, a small nonenzymatic protein. However, it is not known whether nsp10 functions in the regulation of CoV replication fidelity. To test this, we engineered single and double alanine substitution mutations into the genome of murine hepatitis virus (MHV-A59) containing ExoN activity [ExoN(+)] at positions within nsp10 known to disrupt the nsp10-nsp14 interactionin vitro. We show that an nsp10 mutant, R80A/E82A-ExoN(+), was five to ten times more sensitive to treatment with the RNA mutagen 5-fluorouracil (5-FU) than wild-type (WT)-ExoN(+), suggestive of decreased replication fidelity. This decreased-fidelity phenotype was confirmed using two additional nucleoside analogs, 5-azacytidine and ribavirin. R80A/E82A-ExoN(+) reached a peak titer similar to and demonstrated RNA synthesis kinetics comparable to those seen with WT-ExoN(+). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN(−) relative to MHV-ExoN(−), indicating that the decreased-fidelity phenotype of R80A/E82A-ExoN(−) is linked to the presence of ExoN activity. Our results demonstrate that nsp10 is important for CoV replication fidelity and support the hypothesis that nsp10 functions to regulate nsp14-ExoN activity during virus replication.IMPORTANCEThe adaptive capacity of CoVs, as well as all other RNA viruses, is partially attributed to the presence of extensive population genetic diversity. However, decreased fidelity is detrimental to CoV replication and virulence; mutant CoVs with decreased replication fidelity are attenuated and more sensitive to inhibition by RNA mutagens. Thus, identifying the viral protein determinants of CoV fidelity is important for understanding CoV replication, pathogenesis, and virulence. In this report, we show that nsp10, a small, nonenzymatic viral protein, contributes to CoV replication fidelity. Our data support the hypothesis that CoVs have evolved multiple proteins, in addition to nsp14-ExoN, that are responsible for maintaining the integrity of the largest known RNA genomes.

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Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived PeptideIn VitroandIn VivoTo Reduce Replication and Pathogenesis

Journal of Virology ◽

10.1128/jvi.00948-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8416-8427 ◽

Cited By ~ 50

Author(s):

Yi Wang ◽

Ying Sun ◽

Andong Wu ◽

Shan Xu ◽

Ruangang Pan ◽

...

Keyword(s):

Viral Replication ◽

Virus Replication ◽

Host Cells ◽

Common Mechanism ◽

Type I ◽

Link Type ◽

Mtase Activity ◽

Stimulation Of

ABSTRACTThe 5′ cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases (2′-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5′ end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2′-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2′-O-MTase activity (E. Decroly et al., J Virol 82:8071–8084, 2008,http://dx.doi.org/10.1128/JVI.00407-08; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010,http://dx.doi.org/10.1371/journal.ppat.1000863; E. Decroly et al., PLoS Pathog 7:e1002059, 2011,http://dx.doi.org/10.1371/journal.ppat.1002059; Y. Chen et al., PLoS Pathog 7:e1002294, 2011,http://dx.doi.org/10.1371/journal.ppat.1002294) . In this study, we demonstrate that stimulation of nsp16 2′-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2′-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effectsin vivoin MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2′-O-MTase activity can be targetedin vitroandin vivo.IMPORTANCECoronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2′-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2′-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2′-O-MTase activity of different coronavirusesin vitroand viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2′-O-MTase of coronaviruses.

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Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis

Journal of Virology ◽

10.1128/jvi.75.6.2803-2809.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2803-2809 ◽

Cited By ~ 21

Author(s):

Andreas F. Kolb ◽

Lecia Pewe ◽

John Webster ◽

Stanley Perlman ◽

C. Bruce A. Whitelaw ◽

...

Keyword(s):

Transgenic Mice ◽

Neutralizing Antibodies ◽

Defense Mechanism ◽

Viral Infections ◽

Hepatitis Virus ◽

Recombinant Antibody ◽

Lethal Dose ◽

Neutralizing Antibody ◽

Murine Hepatitis Virus

ABSTRACT Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.

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In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination.

The Journal of Cell Biology ◽

10.1083/jcb.111.3.1183 ◽

1990 ◽

Vol 111 (3) ◽

pp. 1183-1195 ◽

Cited By ~ 80

Author(s):

R Armstrong ◽

V L Friedrich ◽

K V Holmes ◽

M Dubois-Dalcq

Keyword(s):

Growth Factor ◽

Glial Cells ◽

Demyelinating Disease ◽

Hepatitis Virus ◽

Relative Proportion ◽

Biological Properties ◽

Murine Hepatitis Virus ◽

Adult Mice ◽

Igf I

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.

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Transcriptional role of p53 in interferon-mediated antiviral immunity

Journal of Experimental Medicine ◽

10.1084/jem.20080383 ◽

2008 ◽

Vol 205 (8) ◽

pp. 1929-1938 ◽

Cited By ~ 130

Author(s):

César Muñoz-Fontela ◽

Salvador Macip ◽

Luis Martínez-Sobrido ◽

Lauren Brown ◽

Joseph Ashour ◽

...

Keyword(s):

Tumor Suppressor ◽

Viral Replication ◽

Viral Infections ◽

Suppressor Gene ◽

Central Component ◽

Type I ◽

Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

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Effect of eicosanoids on induction of procoagulant activity by murine hepatitis virus strain 3 in vitro

Prostaglandins ◽

10.1016/0090-6980(91)90013-6 ◽

1991 ◽

Vol 42 (6) ◽

pp. 501-513 ◽

Cited By ~ 6

Author(s):

S.W. Chung ◽

S.B. Sinclair ◽

L.S. Fung ◽

E.H. Cole ◽

G.A. Levy

Keyword(s):

Virus Strain ◽

Hepatitis Virus ◽

Procoagulant Activity ◽

Murine Hepatitis Virus

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STAT2-Dependent Immune Responses Ensure Host Survival despite the Presence of a Potent Viral Antagonist

Journal of Virology ◽

10.1128/jvi.00296-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 10

Author(s):

Vu Thuy Khanh Le-Trilling ◽

Kerstin Wohlgemuth ◽

Meike U. Rückborn ◽

Andreja Jagnjic ◽

Fabienne Maaßen ◽

...

Keyword(s):

Immune Responses ◽

Viral Replication ◽

Mutual Influence ◽

Type I ◽

Mcmv Infection ◽

General Importance ◽

Interferon Antagonism ◽

Deficient Mice

ABSTRACTA pathogen encounter induces interferons, which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, the host and pathogens are situated in a continuous arms race which shapes host evolution toward optimized immune responses and the pathogens toward enhanced immune-evasive properties. Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2, which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lackingM27and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pairin vitroandin vivo. In contrast to wild-type (wt) MCMV, ΔM27 mutant MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g., liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection. Taken together, the results of our study reveal the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary standoff situation with fatal consequences when the equilibrium is disturbed.IMPORTANCEThe host limits viral replication by the use of interferons (IFNs), which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g., cytomegaloviruses, Zika virus, dengue virus, and several paramyxoviruses). We analyzed infections caused by MCMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate its importance for the host and the virusin vitroandin vivo. The inability to counteract STAT2 directly translates into exaggerated IFN susceptibilityin vitroand pronounced attenuationin vivo. Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.

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Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

Journal of Virology ◽

10.1128/jvi.00338-15 ◽

2015 ◽

Vol 89 (9) ◽

pp. 4907-4917 ◽

Cited By ~ 28

Author(s):

Anna M. Mielech ◽

Xufang Deng ◽

Yafang Chen ◽

Eveline Kindler ◽

Dorthea L. Wheeler ◽

...

Keyword(s):

Viral Replication ◽

Protease Activity ◽

Reverse Genetics ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Viral Pathogenesis ◽

Wild Type ◽

Viral Protease ◽

Novel Approach ◽

Protease Stability

ABSTRACTUbiquitin-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.

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