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 Proofreading-deficient coronaviruses adapt over long-term passage for increased fidelity and fitness without reversion of exoribonuclease-inactivating mutations

2017 ◽  
Author(s):  
Kevin W. Graepel ◽  
Xiaotao Lu ◽  
James Brett Case ◽  
Nicole R. Sexton ◽  
Everett Clinton Smith ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Hepatitis Virus ◽  
Nucleoside Analogs ◽  
High Fidelity ◽  
Murine Hepatitis Virus ◽  
Replication Fidelity ◽  
Competitive Fitness ◽  
Rna Genome ◽  
Inactivating Mutations

ABSTRACTThe coronavirus (CoV) RNA genome is the largest among single-stranded positive sense RNA viruses. CoVs encode a proofreading 3′→5′exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in SARS-CoV and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold decreased fidelity, and increased susceptibility to nucleoside analogs. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with WT-MHV. Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication, reduced susceptibility to nucleoside analogs, and increased competitive fitness. However, passage did not select for complete or partial reversion at the ExoN-inactivating mutations. We identified novel amino acid changes within the RNA-dependent RNA polymerase (nsp12-RdRp) and nsp14 of MHV-ExoN(-) P250 that partially account for the observed changes in replication, susceptibility to nucleoside analogs, and competitive fitness observed in the passaged virus population, indicating that additional determinants can compensate for the activities of nsp14-ExoN. Our results suggest that while selection favors restoration of replication fidelity in ExoN(-) CoVs, there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and identify additional candidate proteins that may regulate CoV replication fidelity.IMPORTANCEUnique among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] are either non-viable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we show that ExoN(-) murine hepatitis virus can adapt over long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogs that is only partially explained by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogs is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

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

2007 ◽  
Vol 81 (22) ◽  
pp. 12135-12144 ◽  
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.

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

2016 ◽  
Vol 90 (16) ◽  
pp. 7248-7256 ◽  
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.

scholarly journals Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

2016 ◽  
Vol 90 (16) ◽  
pp. 7415-7428 ◽  
Author(s):  
Nicole R. Sexton ◽  
Everett Clinton Smith ◽  
Hervé Blanc ◽  
Marco Vignuzzi ◽  
Olve B. Peersen ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Fold Increase ◽  
Rna Polymerases ◽  
Coxsackie Virus ◽  
Murine Hepatitis Virus ◽  
Replication Fidelity ◽  
Replicase Proteins ◽  
Positive Sense

ABSTRACTPositive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex.IMPORTANCERNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

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

2001 ◽  
Vol 75 (6) ◽  
pp. 2803-2809 ◽  
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.

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

1990 ◽  
Vol 111 (3) ◽  
pp. 1183-1195 ◽  
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.

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

1991 ◽  
Vol 42 (6) ◽  
pp. 501-513 ◽  
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

scholarly journals IN VITRO INTERACTION OF MOUSE HEPATITIS VIRUS AND MACROPHAGES FROM GENETICALLY RESISTANT MICE

1970 ◽  
Vol 131 (4) ◽  
pp. 843-850 ◽  
Author(s):  
Ilan Shif ◽  
Frederik B. Bang
Keyword(s):  
Virus Replication ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Peritoneal Macrophages ◽  
Slow Inactivation ◽  
Virus Particles ◽  
C3h Mice ◽  
The Difference ◽  
In Vitro Interaction

Peritoneal macrophages from genetically resistant C3H mice and genetically susceptible Princeton (PRI) mice adsorbed the MHV (PRI) strain of mouse hepatitis virus equally well. The difference between the permissive cells and the nonpermissive ones seems to reside in the ability of the former to "eclipse" the virus and, subsequently, support virus replication. C3H cells exposed to low multiplicities of the virus remained intact with no demonstrable viral replication. Virus, taken up by the resistant cells, was protected from heat and underwent slow inactivation while few or no virus particles were released into the medium.

scholarly journals Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): Studies in vivo and in vitro

Glia ◽  
1989 ◽  
Vol 2 (2) ◽  
pp. 85-93 ◽  
Author(s):  
M.F. van Berlo ◽  
R. Warringa ◽  
G. Wolswijk ◽  
M. Lopes-Cardozo
Keyword(s):  
Mouse Brain ◽  
Hepatitis Virus ◽  
Brain Cells ◽  
Murine Hepatitis Virus

scholarly journals Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

2015 ◽  
Vol 90 (5) ◽  
pp. 2616-2627 ◽  
Author(s):  
Natasha L. Tilston-Lunel ◽  
Gustavo Olszanski Acrani ◽  
Richard E. Randall ◽  
Richard M. Elliott
Keyword(s):  
South America ◽  
Virus Replication ◽  
Nonstructural Protein ◽  
Urban Expansion ◽  
Febrile Illness ◽  
Wild Type Virus ◽  
Human Pathogen ◽  
Coding Region ◽  
Successful Recovery

ABSTRACTOropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried outin vitrodemonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested.IMPORTANCEOropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen.

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