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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

Selection for West Nile virus populations with decreased susceptibility to ribavirin.A. Ribavirin susceptibility, measured as mean log10 pfu/ml reduction in viral titer +/- SD resulting from 50uM ribavirin treatment for 72h on Hela cell culture relative to untreated controls, following passage of parallel series of ribavirin-treated WNV-IC designated as lineage I (black) and lineage II (grey). B. Ribavirin susceptibility in selected clonal populations. Clonal populations were created with lineage I from 20 plaques isolations (pp) and strains with the highest levels of resistance (WNV pp3/9) were chosen for further characterization.
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ppat.1005009.g001: Selection for West Nile virus populations with decreased susceptibility to ribavirin.A. Ribavirin susceptibility, measured as mean log10 pfu/ml reduction in viral titer +/- SD resulting from 50uM ribavirin treatment for 72h on Hela cell culture relative to untreated controls, following passage of parallel series of ribavirin-treated WNV-IC designated as lineage I (black) and lineage II (grey). B. Ribavirin susceptibility in selected clonal populations. Clonal populations were created with lineage I from 20 plaques isolations (pp) and strains with the highest levels of resistance (WNV pp3/9) were chosen for further characterization.

Mentions: Passaging in the presence of the antiviral ribavirin was used to select for WNV variants with decreased susceptibility and putative alterations to polymerase fidelity. Ribavirin susceptibility, as measured by reduction in viral titer following treatment, was monitored throughout the passage series and again assessed following the completion of passage 6 (Fig 1A). Significantly lower reductions in viral titer relative to WNV-IC controls were measured in both lineage I and II following passage 5 and 6 (t-test, df = 5, p<0.05), such that lineage I viral titer decreased just 1.4-fold following ribavirin treatment after 6 passages, as compared to a greater than 25-fold titer reduction measured for WNV-IC. In order to select for clonal strains with decreased mutagen susceptibility, ribavirin sensitivity was assessed for individual biological clones isolated from the lineage I passed virus and strains with the highest levels of resistance (WNV pp3, pp9; Fig 1B) were chosen for further characterization.


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)

Selection for West Nile virus populations with decreased susceptibility to ribavirin.A. Ribavirin susceptibility, measured as mean log10 pfu/ml reduction in viral titer +/- SD resulting from 50uM ribavirin treatment for 72h on Hela cell culture relative to untreated controls, following passage of parallel series of ribavirin-treated WNV-IC designated as lineage I (black) and lineage II (grey). B. Ribavirin susceptibility in selected clonal populations. Clonal populations were created with lineage I from 20 plaques isolations (pp) and strains with the highest levels of resistance (WNV pp3/9) were chosen for further characterization.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005009.g001: Selection for West Nile virus populations with decreased susceptibility to ribavirin.A. Ribavirin susceptibility, measured as mean log10 pfu/ml reduction in viral titer +/- SD resulting from 50uM ribavirin treatment for 72h on Hela cell culture relative to untreated controls, following passage of parallel series of ribavirin-treated WNV-IC designated as lineage I (black) and lineage II (grey). B. Ribavirin susceptibility in selected clonal populations. Clonal populations were created with lineage I from 20 plaques isolations (pp) and strains with the highest levels of resistance (WNV pp3/9) were chosen for further characterization.
Mentions: Passaging in the presence of the antiviral ribavirin was used to select for WNV variants with decreased susceptibility and putative alterations to polymerase fidelity. Ribavirin susceptibility, as measured by reduction in viral titer following treatment, was monitored throughout the passage series and again assessed following the completion of passage 6 (Fig 1A). Significantly lower reductions in viral titer relative to WNV-IC controls were measured in both lineage I and II following passage 5 and 6 (t-test, df = 5, p<0.05), such that lineage I viral titer decreased just 1.4-fold following ribavirin treatment after 6 passages, as compared to a greater than 25-fold titer reduction measured for WNV-IC. In order to select for clonal strains with decreased mutagen susceptibility, ribavirin sensitivity was assessed for individual biological clones isolated from the lineage I passed virus and strains with the highest levels of resistance (WNV pp3, pp9; Fig 1B) were chosen for further characterization.

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