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Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, Scherbakova S, Graham RL, Baric RS, Stockwell TB, Spiro DJ, Denison MR - PLoS Pathog. (2010)

Bottom Line: However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage.Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV.The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Microbiology and Immunology and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

ABSTRACT
Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

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Growth analysis of viral replication.Growth comparisons of SARS-WT and S-ExoN1 viruses (A), and of multiple S-ExoN1 clones and S-ExoN1 population virus (B). (A) Vero cells were infected with SARS-WT P4 c21 and S-ExoN1 P4 c53 viruses at an MOI of 0.1 PFU/cell. (B) Vero cells were infected with P1 population stock of S-ExoN1 or S-ExoN1 P4 clones (c53, c62, c63, c64, and c65) at an MOI of 0.01 PFU/cell. Samples of culture medium were obtained at 1, 4, 8, 12, 16, 20, 24, 30, 36, and 48 h p.i., and viral titers were determined by plaque assay. Mean titers and standard deviations from triplicate infection series are indicated for each time point.
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ppat-1000896-g003: Growth analysis of viral replication.Growth comparisons of SARS-WT and S-ExoN1 viruses (A), and of multiple S-ExoN1 clones and S-ExoN1 population virus (B). (A) Vero cells were infected with SARS-WT P4 c21 and S-ExoN1 P4 c53 viruses at an MOI of 0.1 PFU/cell. (B) Vero cells were infected with P1 population stock of S-ExoN1 or S-ExoN1 P4 clones (c53, c62, c63, c64, and c65) at an MOI of 0.01 PFU/cell. Samples of culture medium were obtained at 1, 4, 8, 12, 16, 20, 24, 30, 36, and 48 h p.i., and viral titers were determined by plaque assay. Mean titers and standard deviations from triplicate infection series are indicated for each time point.

Mentions: To determine whether substitution of ExoN active-site residues altered viral replication, we performed growth assays in Vero cells using P4 stocks of SARS-WT plaque clones 14 and 21 (c14 and c21) and S-ExoN1 c53 at a multiplicity of infection (MOI) of 0.1 plaque-forming units (PFU) per cell. Growth kinetics of S-ExoN1 and both SARS-WT clones were identical for the first 20 h, after which S-ExoN1 exhibited reduced viral titers compared to SARS-WT. SARS-WT and S-ExoN1 achieved peak viral titers at 30–36 hpi, although this was reduced by 4-fold for S-ExoN1 (Figure 3A). Both SARS-WT clones showed identical growth throughout the experiment (data not shown). These results indicate that inactivation of ExoN results in impaired replication of SARS-CoV.


Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, Scherbakova S, Graham RL, Baric RS, Stockwell TB, Spiro DJ, Denison MR - PLoS Pathog. (2010)

Growth analysis of viral replication.Growth comparisons of SARS-WT and S-ExoN1 viruses (A), and of multiple S-ExoN1 clones and S-ExoN1 population virus (B). (A) Vero cells were infected with SARS-WT P4 c21 and S-ExoN1 P4 c53 viruses at an MOI of 0.1 PFU/cell. (B) Vero cells were infected with P1 population stock of S-ExoN1 or S-ExoN1 P4 clones (c53, c62, c63, c64, and c65) at an MOI of 0.01 PFU/cell. Samples of culture medium were obtained at 1, 4, 8, 12, 16, 20, 24, 30, 36, and 48 h p.i., and viral titers were determined by plaque assay. Mean titers and standard deviations from triplicate infection series are indicated for each time point.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865531&req=5

ppat-1000896-g003: Growth analysis of viral replication.Growth comparisons of SARS-WT and S-ExoN1 viruses (A), and of multiple S-ExoN1 clones and S-ExoN1 population virus (B). (A) Vero cells were infected with SARS-WT P4 c21 and S-ExoN1 P4 c53 viruses at an MOI of 0.1 PFU/cell. (B) Vero cells were infected with P1 population stock of S-ExoN1 or S-ExoN1 P4 clones (c53, c62, c63, c64, and c65) at an MOI of 0.01 PFU/cell. Samples of culture medium were obtained at 1, 4, 8, 12, 16, 20, 24, 30, 36, and 48 h p.i., and viral titers were determined by plaque assay. Mean titers and standard deviations from triplicate infection series are indicated for each time point.
Mentions: To determine whether substitution of ExoN active-site residues altered viral replication, we performed growth assays in Vero cells using P4 stocks of SARS-WT plaque clones 14 and 21 (c14 and c21) and S-ExoN1 c53 at a multiplicity of infection (MOI) of 0.1 plaque-forming units (PFU) per cell. Growth kinetics of S-ExoN1 and both SARS-WT clones were identical for the first 20 h, after which S-ExoN1 exhibited reduced viral titers compared to SARS-WT. SARS-WT and S-ExoN1 achieved peak viral titers at 30–36 hpi, although this was reduced by 4-fold for S-ExoN1 (Figure 3A). Both SARS-WT clones showed identical growth throughout the experiment (data not shown). These results indicate that inactivation of ExoN results in impaired replication of SARS-CoV.

Bottom Line: However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage.Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV.The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Microbiology and Immunology and Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

ABSTRACT
Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

Show MeSH
Related in: MedlinePlus