scholarly journals The coronavirus proofreading exoribonuclease mediates extensive viral recombination

2020 ◽  
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.

scholarly journals The coronavirus proofreading exoribonuclease mediates extensive viral recombination

2021 ◽  
Vol 17 (1) ◽  
pp. e1009226
Author(s):  
Jennifer Gribble ◽  
Laura J. Stevens ◽  
Maria L. Agostini ◽  
Jordan Anderson-Daniels ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Rna Synthesis ◽  
Recombination Frequency ◽  
Hepatitis Virus ◽  
Nucleoside Analogues ◽  
High Fidelity ◽  
Rna Recombination ◽  
Murine Hepatitis Virus ◽  
Critical Function ◽  
Viral Recombination

Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.

scholarly journals A New Cistron in the Murine Hepatitis Virus Replicase Gene

2010 ◽  
Vol 84 (19) ◽  
pp. 10148-10158 ◽  
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.

scholarly journals Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function

2009 ◽  
Vol 84 (1) ◽  
pp. 280-290 ◽  
Author(s):  
Mark J. Gadlage ◽  
Jennifer S. Sparks ◽  
Dia C. Beachboard ◽  
Reagan G. Cox ◽  
Joshua D. Doyle ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Complex Function ◽  
Microscopic Analysis ◽  
Murine Hepatitis Virus ◽  
Electron Microscopic ◽  
Virus Growth ◽  
Positive Strand Rna

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.

scholarly journals Inhibiting ACSL1 related ferroptosis restrains MHV-A59 infection

2021 ◽  
Author(s):  
Huawei Xia ◽  
Zeming Zhang ◽  
Fuping You
Keyword(s):  
Hepatitis Virus ◽  
Murine Hepatitis Virus ◽  
Viral Propagation ◽  
Triacsin C ◽  
Representative Model ◽  
Infected Cells ◽  
Coronavirus Infection ◽  
Syncytia Formation ◽  
In Vivo Administration

Murine hepatitis virus strain A59 (MHV-A59) belongs to the β-coronavirus and is considered as a representative model for studying coronavirus infection. MHV-A59 was shown to induce pyroptosis, apoptosis and necroptosis of infected cells, especially the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59, is unknown. Here, we demonstrate inhibiting ferroptosis suppresses MHV-A59 infection. MHV-A59 infection upregulates the expression of Acsl1, a novel ferroptosis inducer. MHV-A59 upregulates Acsl1 expression depending on the NF-kB activation, which is TLR4-independent. Ferroptosis inhibitor inhibits viral propagation, inflammatory cytokines release and MHV-A59 infection induced cell syncytia formation. ACSL1 inhibitor Triacsin C suppresses MHV-A59 infection induced syncytia formation and viral propagation. In vivo administration of liproxstatin-1 ameliorates lung inflammation and tissue injuries caused by MHV-A59 infection. Collectively, these results indicate that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serves as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.

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

2006 ◽  
Vol 80 (16) ◽  
pp. 7894-7901 ◽  
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.

scholarly journals Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches

2009 ◽  
Vol 83 (13) ◽  
pp. 6957-6962 ◽  
Author(s):  
Surendranath Baliji ◽  
Stephen A. Cammer ◽  
Bruno Sobral ◽  
Susan C. Baker
Keyword(s):  
Viral Replication ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Nonstructural Proteins ◽  
Hydrophobic Domain ◽  
Transmembrane Topology ◽  
Molecular Approaches ◽  
Viral Proteases ◽  
Infected Cells ◽  
Membrane Spanning

ABSTRACT Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.

scholarly journals Mutations in Coronavirus Nonstructural Protein 10 Decrease Virus Replication Fidelity

2015 ◽  
Vol 89 (12) ◽  
pp. 6418-6426 ◽  
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.

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

2007 ◽  
Vol 81 (12) ◽  
pp. 6700-6708 ◽  
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.

scholarly journals Manipulation of the unfolded protein response: A pharmacological strategy against coronavirus infection

2021 ◽  
Vol 17 (6) ◽  
pp. e1009644
Author(s):  
Liliana Echavarría-Consuegra ◽  
Georgia M. Cook ◽  
Idoia Busnadiego ◽  
Charlotte Lefèvre ◽  
Sarah Keep ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Hepatitis Virus ◽  
Ribosome Profiling ◽  
Murine Hepatitis Virus ◽  
Unfolded Protein ◽  
Virion Release ◽  
Infected Cells ◽  
Coronavirus Infection ◽  
The Unfolded Protein Response ◽  
Protein Response

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

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