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Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

Van Slyke GA, Arnold JJ, Lugo AJ, Griesemer SB, Moustafa IM, Kramer LD, Cameron CE, Ciota AT - PLoS Pathog. (2015)

Bottom Line: We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity.Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes.Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: The Arbovirus Laboratory, Wadsworth Center, New York State Department of Health, Slingerlands, New York, United States of America.

ABSTRACT
High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

No MeSH data available.


Related in: MedlinePlus

Strain-specific differences in mutational spectra breadth and composition.WNV mutant swarm characterization by deep-sequencing of nucleotides 1312–3261 (ENV/NS1) following 72h growth on mosquito cell culture. A. Mean distribution of genomic diversity (single nucleotide polymorphisms [SNP]) of 2 replicates of WNV-IC across the sequenced region. The distribution of mutations was similar among strains. B. Mean number of minority SNPs identified for individual WNV strains at frequencies greater than 0.5%. C. Distribution of substitution types among WNV strains.
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ppat.1005009.g004: Strain-specific differences in mutational spectra breadth and composition.WNV mutant swarm characterization by deep-sequencing of nucleotides 1312–3261 (ENV/NS1) following 72h growth on mosquito cell culture. A. Mean distribution of genomic diversity (single nucleotide polymorphisms [SNP]) of 2 replicates of WNV-IC across the sequenced region. The distribution of mutations was similar among strains. B. Mean number of minority SNPs identified for individual WNV strains at frequencies greater than 0.5%. C. Distribution of substitution types among WNV strains.

Mentions: In order to test the hypothesis that mutagen resistance corresponded to alterations in mutation rate, deep-sequencing was used to quantify accumulation of unique SNPs following a single passage on mosquito cell culture (Fig 4A). Levels of WNV RNA for both WNV-IC and mutant strains were statistically similar for all samples chosen for sequencing (~9.0 log10 WNV copies/ml), suggesting differences in replication were not likely to account for differences in the number of mutations accumulated. Assays were completed in duplicate for each WNV strain and sequencing results identified fewer SNPs in all mutant strains relative to WNV-IC, with the exception of the methyltransferase mutant, WNV T248I, for which the number of unique SNPs identified was approximately 2.5 fold higher than WNV-IC. The RdRp double mutant, WNV V793I/G806R, showed the fewest number of unique SNPs; approximately 2.5 fold lower than WNV-IC (Fig 4B). Mutations were distributed throughout the sequenced regions for all WNV strains, yet mutant swarm composition varied significantly among strains (Fig 4C). Specifically, transition to transversion ratios were ~2:1 for WNV-IC and WNV T248I, yet <1 for WNV V793I/G806R. Decreased mutation of WNV V793I/G806R relative to WNV-IC resulted primarily from lack of U to C and G to A mutations, which accounted for 11/28 mutations for WNV-IC and 0/12 mutations for WNV V793I/G806R (chi-squared, p = 0.011). Increased mutation of WNV T248I, on the other hand, resulted primarily from A misincorporations, which accounted for 33/62 mutations for WNV T248I, and just 7/28 mutations of WNV-IC (chi-squared, p = 0.045). Approximately the same number of U to A mutations were identified for WNV V793I/G806R as WNV T248I (4 vs 3), yet no G to A mutations were identified for WNV V793I/G806R, in stark contrast to the 20 identified for WNV T248I (Fig 4C). These results demonstrate strain-specific differences in the misincorporation of different nucleotides and, more specifically, in the propensity for particular mispairs, suggesting mutation frequencies may be dependent on both replication complex and template sequences and/or context.


Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

Van Slyke GA, Arnold JJ, Lugo AJ, Griesemer SB, Moustafa IM, Kramer LD, Cameron CE, Ciota AT - PLoS Pathog. (2015)

Strain-specific differences in mutational spectra breadth and composition.WNV mutant swarm characterization by deep-sequencing of nucleotides 1312–3261 (ENV/NS1) following 72h growth on mosquito cell culture. A. Mean distribution of genomic diversity (single nucleotide polymorphisms [SNP]) of 2 replicates of WNV-IC across the sequenced region. The distribution of mutations was similar among strains. B. Mean number of minority SNPs identified for individual WNV strains at frequencies greater than 0.5%. C. Distribution of substitution types among WNV strains.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482725&req=5

ppat.1005009.g004: Strain-specific differences in mutational spectra breadth and composition.WNV mutant swarm characterization by deep-sequencing of nucleotides 1312–3261 (ENV/NS1) following 72h growth on mosquito cell culture. A. Mean distribution of genomic diversity (single nucleotide polymorphisms [SNP]) of 2 replicates of WNV-IC across the sequenced region. The distribution of mutations was similar among strains. B. Mean number of minority SNPs identified for individual WNV strains at frequencies greater than 0.5%. C. Distribution of substitution types among WNV strains.
Mentions: In order to test the hypothesis that mutagen resistance corresponded to alterations in mutation rate, deep-sequencing was used to quantify accumulation of unique SNPs following a single passage on mosquito cell culture (Fig 4A). Levels of WNV RNA for both WNV-IC and mutant strains were statistically similar for all samples chosen for sequencing (~9.0 log10 WNV copies/ml), suggesting differences in replication were not likely to account for differences in the number of mutations accumulated. Assays were completed in duplicate for each WNV strain and sequencing results identified fewer SNPs in all mutant strains relative to WNV-IC, with the exception of the methyltransferase mutant, WNV T248I, for which the number of unique SNPs identified was approximately 2.5 fold higher than WNV-IC. The RdRp double mutant, WNV V793I/G806R, showed the fewest number of unique SNPs; approximately 2.5 fold lower than WNV-IC (Fig 4B). Mutations were distributed throughout the sequenced regions for all WNV strains, yet mutant swarm composition varied significantly among strains (Fig 4C). Specifically, transition to transversion ratios were ~2:1 for WNV-IC and WNV T248I, yet <1 for WNV V793I/G806R. Decreased mutation of WNV V793I/G806R relative to WNV-IC resulted primarily from lack of U to C and G to A mutations, which accounted for 11/28 mutations for WNV-IC and 0/12 mutations for WNV V793I/G806R (chi-squared, p = 0.011). Increased mutation of WNV T248I, on the other hand, resulted primarily from A misincorporations, which accounted for 33/62 mutations for WNV T248I, and just 7/28 mutations of WNV-IC (chi-squared, p = 0.045). Approximately the same number of U to A mutations were identified for WNV V793I/G806R as WNV T248I (4 vs 3), yet no G to A mutations were identified for WNV V793I/G806R, in stark contrast to the 20 identified for WNV T248I (Fig 4C). These results demonstrate strain-specific differences in the misincorporation of different nucleotides and, more specifically, in the propensity for particular mispairs, suggesting mutation frequencies may be dependent on both replication complex and template sequences and/or context.

Bottom Line: We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity.Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes.Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: The Arbovirus Laboratory, Wadsworth Center, New York State Department of Health, Slingerlands, New York, United States of America.

ABSTRACT
High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

No MeSH data available.


Related in: MedlinePlus