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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

Specific infectivity of WNV replication complex mutants.WNV RNA (viral particles) and infectious virus (pfu) were quantified following 1-step growth on mosquito (CxT) or vertebrate (BHK) cell culture and specific infectivity (particle/pfu) was compared among WNV-IC, WNV V793I/G806R and WNV T248I. Graphs represent means +/- standard deviation. Significantly decreased infectivity (*) was measured for WNV T248I relative to WNV-IC and WNV V793I/G806R (t-test, df = 4, p = 0.0028).
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ppat.1005009.g007: Specific infectivity of WNV replication complex mutants.WNV RNA (viral particles) and infectious virus (pfu) were quantified following 1-step growth on mosquito (CxT) or vertebrate (BHK) cell culture and specific infectivity (particle/pfu) was compared among WNV-IC, WNV V793I/G806R and WNV T248I. Graphs represent means +/- standard deviation. Significantly decreased infectivity (*) was measured for WNV T248I relative to WNV-IC and WNV V793I/G806R (t-test, df = 4, p = 0.0028).

Mentions: Comparison of one-step and multi-step growth kinetics of WNV mutants to WNV-IC in vertebrate (BHK) and invertebrate (CxT) cell lines demonstrates host-specific effects of replication complex mutations (Fig 6). No differences in overall kinetics (repeated measures ANOVA, F = 0.14, df = 6, p = 0.98) or viral titers at individual time points (t-test, p>0.05) were measured when comparing WNV mutants to WNV-IC on vertebrate cell culture, while significant differences in viral kinetics were measured in mosquito cell culture at both MOIs for all replication complex mutants relative to WNV-IC (repeated measures ANOVA, F = 12.83, df = 6, p<0.0001). Specifically, consistently lower viral titers were measured for all RdRp and Mtase mutants in mosquito cells relative to WNV-IC (Tukey’s multiple comparison test, p<0.05), and kinetics were similar among mutants with the exception of WNV V793I, which despite having significantly lower titers than WNV-IC had modestly higher titers relative to other mutants (Fig 6). In addition, WNV RNA was quantified with qRT-PCR following one-step growth and particle/pfu ratios were quantified and compared among WNV-IC, WNV V793I/G806R and WNV T248I in order to assess the relationship between fidelity and infectivity. Specific infectivity was elevated in mosquito cells as compared to vertebrate cells for all WNV strains (Fig 7). Trends measured with infectivity are consistent with identified fidelity differences in both cell lines, with the highest infectivity measured with the RdRp double mutant WNV V793I/G806R and the lowest infectivity measured with the Mtase mutant WNV T248I, yet differences were only significant relative to WNV-IC for WNV T248I in BHK cells (t-test, df = 4, p = 0.0028; Fig 7). These differences do not therefore account for differences in growth kinetics identified among WNV strains in CxT cells.


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)

Specific infectivity of WNV replication complex mutants.WNV RNA (viral particles) and infectious virus (pfu) were quantified following 1-step growth on mosquito (CxT) or vertebrate (BHK) cell culture and specific infectivity (particle/pfu) was compared among WNV-IC, WNV V793I/G806R and WNV T248I. Graphs represent means +/- standard deviation. Significantly decreased infectivity (*) was measured for WNV T248I relative to WNV-IC and WNV V793I/G806R (t-test, df = 4, p = 0.0028).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005009.g007: Specific infectivity of WNV replication complex mutants.WNV RNA (viral particles) and infectious virus (pfu) were quantified following 1-step growth on mosquito (CxT) or vertebrate (BHK) cell culture and specific infectivity (particle/pfu) was compared among WNV-IC, WNV V793I/G806R and WNV T248I. Graphs represent means +/- standard deviation. Significantly decreased infectivity (*) was measured for WNV T248I relative to WNV-IC and WNV V793I/G806R (t-test, df = 4, p = 0.0028).
Mentions: Comparison of one-step and multi-step growth kinetics of WNV mutants to WNV-IC in vertebrate (BHK) and invertebrate (CxT) cell lines demonstrates host-specific effects of replication complex mutations (Fig 6). No differences in overall kinetics (repeated measures ANOVA, F = 0.14, df = 6, p = 0.98) or viral titers at individual time points (t-test, p>0.05) were measured when comparing WNV mutants to WNV-IC on vertebrate cell culture, while significant differences in viral kinetics were measured in mosquito cell culture at both MOIs for all replication complex mutants relative to WNV-IC (repeated measures ANOVA, F = 12.83, df = 6, p<0.0001). Specifically, consistently lower viral titers were measured for all RdRp and Mtase mutants in mosquito cells relative to WNV-IC (Tukey’s multiple comparison test, p<0.05), and kinetics were similar among mutants with the exception of WNV V793I, which despite having significantly lower titers than WNV-IC had modestly higher titers relative to other mutants (Fig 6). In addition, WNV RNA was quantified with qRT-PCR following one-step growth and particle/pfu ratios were quantified and compared among WNV-IC, WNV V793I/G806R and WNV T248I in order to assess the relationship between fidelity and infectivity. Specific infectivity was elevated in mosquito cells as compared to vertebrate cells for all WNV strains (Fig 7). Trends measured with infectivity are consistent with identified fidelity differences in both cell lines, with the highest infectivity measured with the RdRp double mutant WNV V793I/G806R and the lowest infectivity measured with the Mtase mutant WNV T248I, yet differences were only significant relative to WNV-IC for WNV T248I in BHK cells (t-test, df = 4, p = 0.0028; Fig 7). These differences do not therefore account for differences in growth kinetics identified among WNV strains in CxT cells.

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