1H, 13C, 15N resonance assignments of murine hepatitis virus nonstructural protein 3a

ABSTRACT Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

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Crystal Structure of Nonstructural Protein 10 from the Severe Acute Respiratory Syndrome Coronavirus Reveals a Novel Fold with Two Zinc-Binding Motifs

Journal of Virology ◽

10.1128/jvi.00467-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 7894-7901 ◽

Cited By ~ 76

Author(s):

Jeremiah S. Joseph ◽

Kumar Singh Saikatendu ◽

Vanitha Subramanian ◽

Benjamin W. Neuman ◽

Alexei Brooun ◽

...

Keyword(s):

Crystal Structure ◽

Severe Acute Respiratory Syndrome ◽

Structural Integrity ◽

Rna Binding ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Critical Role ◽

Sequence Specificity ◽

Nucleic Acid Binding ◽

Murine Hepatitis Virus

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 “nonstructural” proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular β-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the “gag-knuckle fold group” of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

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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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Crystal Structure of a Monomeric Form of Severe Acute Respiratory Syndrome Coronavirus Endonuclease nsp15 Suggests a Role for Hexamerization as an Allosteric Switch

Journal of Virology ◽

10.1128/jvi.02817-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6700-6708 ◽

Cited By ~ 42

Author(s):

Jeremiah S. Joseph ◽

Kumar Singh Saikatendu ◽

Vanitha Subramanian ◽

Benjamin W. Neuman ◽

Michael J. Buchmeier ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Active Site ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Full Length ◽

Structural Basis ◽

Monomeric Form ◽

Murine Hepatitis Virus ◽

Catalytic Loop ◽

Intersubunit Interactions

ABSTRACT Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn2+-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 Å. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by ∼120° into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

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The coronavirus proofreading exoribonuclease mediates extensive viral recombination

10.1101/2020.04.23.057786 ◽

2020 ◽

Cited By ~ 8

Author(s):

Jennifer Gribble ◽

Andrea J. Pruijssers ◽

Maria L. Agostini ◽

Jordan Anderson-Daniels ◽

James D. Chappell ◽

...

Keyword(s):

Hepatitis Virus ◽

Nonstructural Protein ◽

Severe Disease ◽

Rna Recombination ◽

Murine Hepatitis Virus ◽

Viral Recombination ◽

Viral Genomes ◽

Genetic Inactivation ◽

Virus Inhibition ◽

Infected Cells

SUMMARYCoronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and generates defective viral genomes (DVGs) of unknown function. However, the determinants and patterns of CoV recombination are unknown. Here, we show that divergent β-CoVs SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV) perform extensive RNA recombination in culture, generating similar patterns of recombination junctions and diverse populations of DVGs and sgmRNAs. We demonstrate that the CoV proofreading nonstructural protein (nsp14) 3’-to-5’ exoribonuclease (nsp14-ExoN) is required for normal CoV recombination and that its genetic inactivation causes significantly decreased frequency and altered patterns of recombination in both infected cells and released virions. Thus, nsp14-ExoN is a key determinant of both high fidelity CoV replication and recombination, and thereby represents a highly-conserved and vulnerable target for virus inhibition and attenuation.

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Organ-Specific Attenuation of Murine Hepatitis Virus Strain A59 by Replacement of Catalytic Residues in the Putative Viral Cyclic Phosphodiesterase ns2

Journal of Virology ◽

10.1128/jvi.02203-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3743-3753 ◽

Cited By ~ 30

Author(s):

Jessica K. Roth-Cross ◽

Helen Stokes ◽

Guohui Chang ◽

Ming Ming Chua ◽

Volker Thiel ◽

...

Keyword(s):

Hepatitis Virus ◽

Nonstructural Protein ◽

Genetic System ◽

Single Amino Acid ◽

Reverse Genetic System ◽

Embryonic Cells ◽

Wild Type ◽

Murine Hepatitis Virus ◽

Recombinant Viruses ◽

Organ Specific

ABSTRACT The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.

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Chimeric Exchange of Coronavirus nsp5 Proteases (3CLpro) Identifies Common and Divergent Regulatory Determinants of Protease Activity

Journal of Virology ◽

10.1128/jvi.02050-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 12611-12618 ◽

Cited By ~ 39

Author(s):

Christopher C. Stobart ◽

Nicole R. Sexton ◽

Havisha Munjal ◽

Xiaotao Lu ◽

Katrina L. Molland ◽

...

Keyword(s):

Hepatitis Virus ◽

Nonstructural Protein ◽

Cell Types ◽

Temperature Sensitive ◽

Cleavage Sites ◽

Wild Type ◽

Murine Hepatitis Virus ◽

Direct Competition ◽

Efficient Replication ◽

Human Coronaviruses

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

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High Fidelity of Murine Hepatitis Virus Replication Is Decreased in nsp14 Exoribonuclease Mutants

Journal of Virology ◽

10.1128/jvi.01296-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12135-12144 ◽

Cited By ~ 151

Author(s):

Lance D. Eckerle ◽

Xiaotao Lu ◽

Steven M. Sperry ◽

Leena Choi ◽

Mark R. Denison

Keyword(s):

Active Site ◽

Rna Viruses ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Critical Role ◽

Wild Type Virus ◽

High Fidelity ◽

Wild Type ◽

Murine Hepatitis Virus ◽

Replication Fidelity

ABSTRACT Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3′-to-5′ exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.

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

Journal of Virology ◽

10.1128/jvi.00542-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7248-7256 ◽

Cited By ~ 21

Author(s):

James Brett Case ◽

Alison W. Ashbrook ◽

Terence S. Dermody ◽

Mark R. Denison

Keyword(s):

Viral Replication ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Rna Stability ◽

Type I ◽

Translation Efficiency ◽

Murine Hepatitis Virus ◽

Mtase Activity ◽

Binding Residues

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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A New Cistron in the Murine Hepatitis Virus Replicase Gene

Journal of Virology ◽

10.1128/jvi.00901-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 10148-10158 ◽

Cited By ~ 15

Author(s):

Helen L. Stokes ◽

Surendranath Baliji ◽

Chang Guo Hui ◽

Stanley G. Sawicki ◽

Susan C. Baker ◽

...

Keyword(s):

Gene Locus ◽

Reverse Genetics ◽

Hepatitis Virus ◽

Nonstructural Protein ◽

Temperature Sensitive ◽

Replicase Gene ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Murine Hepatitis Virus ◽

Infected Cells

ABSTRACT We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33°C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5°C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

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