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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

Decreased mutagen susceptibility of WNV replication complex mutants.RdRp and Mtase mutations identified in WNV populations with the highest ribavirin resistance were reverse engineered into the WNV-IC individually (T248I, V793I and G806R) and in combination (V793I/G806R, T248I/V793I/G806R) and assessed for mutagen resistance on Hela cell culture using either 100uM ribavirin or 50uM 5-fluorouracil (5-FU). Bars depict mean reduction in titer +/- standard deviation following mutagen treatment. Significantly higher viral titers (*t-test, df = 5, p<0.05) were measured for WNV-IC relative to all WNV mutants after 72h treatment with both antivirals.
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ppat.1005009.g003: Decreased mutagen susceptibility of WNV replication complex mutants.RdRp and Mtase mutations identified in WNV populations with the highest ribavirin resistance were reverse engineered into the WNV-IC individually (T248I, V793I and G806R) and in combination (V793I/G806R, T248I/V793I/G806R) and assessed for mutagen resistance on Hela cell culture using either 100uM ribavirin or 50uM 5-fluorouracil (5-FU). Bars depict mean reduction in titer +/- standard deviation following mutagen treatment. Significantly higher viral titers (*t-test, df = 5, p<0.05) were measured for WNV-IC relative to all WNV mutants after 72h treatment with both antivirals.

Mentions: To confirm that these amino acid substitutions independently conferred decreased ribavirin susceptibility, and to assess if this corresponded to generalized mutagen resistance, mutations were engineered independently and in combination into the WNV-IC and susceptibility to both ribavirin and 5-fluorouracil was assessed with WNV mutants. Full-genome sequencing following mutagenesis confirmed the exclusive presence of desired mutations. WNV mutants created included WNV T248I, V793I, G806R, double mutant V793I/G806R, and triple mutant (T248I/V793I/G806R). Mutagen resistance assays demonstrated significantly decreased reduction in titer (susceptibility) relative to untreated controls for all mutant strains as compared to WNV-IC following treatment with both ribavirin and 5-fluorouracil (t-test, df = 6 p<0.05; Fig 3). The highest level of mutagen resistance was measured with the RdRp double mutant, WNV V793I/G806R, for which 2.2 and 4.6-fold mean titer reduction were measured following ribavirin and 5-fluorouracil treatments, respectively, as compared to 55 and 37-fold mean titer reductions measured with WNV-IC (Fig 3).


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)

Decreased mutagen susceptibility of WNV replication complex mutants.RdRp and Mtase mutations identified in WNV populations with the highest ribavirin resistance were reverse engineered into the WNV-IC individually (T248I, V793I and G806R) and in combination (V793I/G806R, T248I/V793I/G806R) and assessed for mutagen resistance on Hela cell culture using either 100uM ribavirin or 50uM 5-fluorouracil (5-FU). Bars depict mean reduction in titer +/- standard deviation following mutagen treatment. Significantly higher viral titers (*t-test, df = 5, p<0.05) were measured for WNV-IC relative to all WNV mutants after 72h treatment with both antivirals.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005009.g003: Decreased mutagen susceptibility of WNV replication complex mutants.RdRp and Mtase mutations identified in WNV populations with the highest ribavirin resistance were reverse engineered into the WNV-IC individually (T248I, V793I and G806R) and in combination (V793I/G806R, T248I/V793I/G806R) and assessed for mutagen resistance on Hela cell culture using either 100uM ribavirin or 50uM 5-fluorouracil (5-FU). Bars depict mean reduction in titer +/- standard deviation following mutagen treatment. Significantly higher viral titers (*t-test, df = 5, p<0.05) were measured for WNV-IC relative to all WNV mutants after 72h treatment with both antivirals.
Mentions: To confirm that these amino acid substitutions independently conferred decreased ribavirin susceptibility, and to assess if this corresponded to generalized mutagen resistance, mutations were engineered independently and in combination into the WNV-IC and susceptibility to both ribavirin and 5-fluorouracil was assessed with WNV mutants. Full-genome sequencing following mutagenesis confirmed the exclusive presence of desired mutations. WNV mutants created included WNV T248I, V793I, G806R, double mutant V793I/G806R, and triple mutant (T248I/V793I/G806R). Mutagen resistance assays demonstrated significantly decreased reduction in titer (susceptibility) relative to untreated controls for all mutant strains as compared to WNV-IC following treatment with both ribavirin and 5-fluorouracil (t-test, df = 6 p<0.05; Fig 3). The highest level of mutagen resistance was measured with the RdRp double mutant, WNV V793I/G806R, for which 2.2 and 4.6-fold mean titer reduction were measured following ribavirin and 5-fluorouracil treatments, respectively, as compared to 55 and 37-fold mean titer reductions measured with WNV-IC (Fig 3).

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