Limits...
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

Location of the mutation sites associated with altered fidelity in WNV NS5.A. Shown are the crystal structures of the N-terminal methyltransferase domain (MTase, colored pink; PDB 2OY0) and the C-terminal polymerase domain (RdRp, colored red; PDB 2HFZ) of the NS5 protein from WNV. The relative positioning of the two domains was based on the crystal structure of the full length NS5 protein of DENV (PDB 4V0R). The protein is represented as ribbon and the identified mutants (T248, V793, and G806) are represented as black spheres. The amino acids V793 and G806 are located in the priming loop that is part of the thumb subdomain. B. The initiation model of WNV RdRp showing the thumb subdomain of the polymerase, rotated 180° compared to the view in A, a 4-mer RNA extracted from the ɸ6-RdRp (PDB 1HI0, green carbon atoms), rNTP modeled at the priming site (P) and the catalytic site (C) based on the complex structure of HCV RdRp (PDB G1X5, yellow carbon atoms), and the active-site aspartates D636 and D669 with the bound catalytic Mg2+ ion. The substitutions, V793I and G806R, are expected to impact the interactions of the nearby residues of the priming loop, including W792, T799, W808, and M809. Disruption of these interactions would affect the dynamics of the priming loop, including the conformational change of W800 that is suggested to be required for stabilizing the initiation complex. C. the MTase and polymerase domains shown in (A) are rendered as pink and red surfaces, respectively, to emphasize the expected large interface between the two domains. Residue T248 is colored black; substitution of this residue by an isoleucine could impact the interface between the two domains of NS5 structure. The figure was prepared using program CHIMERA.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482725&req=5

ppat.1005009.g002: Location of the mutation sites associated with altered fidelity in WNV NS5.A. Shown are the crystal structures of the N-terminal methyltransferase domain (MTase, colored pink; PDB 2OY0) and the C-terminal polymerase domain (RdRp, colored red; PDB 2HFZ) of the NS5 protein from WNV. The relative positioning of the two domains was based on the crystal structure of the full length NS5 protein of DENV (PDB 4V0R). The protein is represented as ribbon and the identified mutants (T248, V793, and G806) are represented as black spheres. The amino acids V793 and G806 are located in the priming loop that is part of the thumb subdomain. B. The initiation model of WNV RdRp showing the thumb subdomain of the polymerase, rotated 180° compared to the view in A, a 4-mer RNA extracted from the ɸ6-RdRp (PDB 1HI0, green carbon atoms), rNTP modeled at the priming site (P) and the catalytic site (C) based on the complex structure of HCV RdRp (PDB G1X5, yellow carbon atoms), and the active-site aspartates D636 and D669 with the bound catalytic Mg2+ ion. The substitutions, V793I and G806R, are expected to impact the interactions of the nearby residues of the priming loop, including W792, T799, W808, and M809. Disruption of these interactions would affect the dynamics of the priming loop, including the conformational change of W800 that is suggested to be required for stabilizing the initiation complex. C. the MTase and polymerase domains shown in (A) are rendered as pink and red surfaces, respectively, to emphasize the expected large interface between the two domains. Residue T248 is colored black; substitution of this residue by an isoleucine could impact the interface between the two domains of NS5 structure. The figure was prepared using program CHIMERA.

Mentions: In order to identify shared WNV amino acid (aa) substitutions associated with mutagen resistance, full-genome sequencing of clonal strains WNV pp3 and WNV pp9 was completed. A total of 15 (pp3) and 18 (pp9) nt substitutions were identified, resulting in 9 and 8 aa substitutions, respectively (Table 2). Of these, 10 nt and 7 aa substitutions were shared. Given the assumption that substitutions outside of the replication complex were more likely to be associated with adaptation to Hela cell culture or drift, shared aa substitution in the WNV RdRp and methyltransferase (Mtase) genes exclusively were chosen for further characterization. These included C8423T, resulting in a threonine to isoleucine change at position 248 of the Mtase, as well as G10057A and G10096A, resulting in valine to isoleucine and glycine to arginine changes at positions 793 and 806 of the RdRp, respectively (Table 2). Mapping of these residues on the known flavivirus RdRp and Mtase structures demonstrates that T248I is located at the C-terminal loop (aa 245–267), which is expected to interact with the RdRp domain, and both V793I and G806R exist in locations outside of the RdRp active site, although are within the priming loop (Fig 2; [39–42]). Both T248 and G806 are conserved among lineage I WNV strains, yet not across lineages or species, while V793 is shared among flaviviruses. No naturally circulating strains were found to possess the identified mutations at these locations.


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)

Location of the mutation sites associated with altered fidelity in WNV NS5.A. Shown are the crystal structures of the N-terminal methyltransferase domain (MTase, colored pink; PDB 2OY0) and the C-terminal polymerase domain (RdRp, colored red; PDB 2HFZ) of the NS5 protein from WNV. The relative positioning of the two domains was based on the crystal structure of the full length NS5 protein of DENV (PDB 4V0R). The protein is represented as ribbon and the identified mutants (T248, V793, and G806) are represented as black spheres. The amino acids V793 and G806 are located in the priming loop that is part of the thumb subdomain. B. The initiation model of WNV RdRp showing the thumb subdomain of the polymerase, rotated 180° compared to the view in A, a 4-mer RNA extracted from the ɸ6-RdRp (PDB 1HI0, green carbon atoms), rNTP modeled at the priming site (P) and the catalytic site (C) based on the complex structure of HCV RdRp (PDB G1X5, yellow carbon atoms), and the active-site aspartates D636 and D669 with the bound catalytic Mg2+ ion. The substitutions, V793I and G806R, are expected to impact the interactions of the nearby residues of the priming loop, including W792, T799, W808, and M809. Disruption of these interactions would affect the dynamics of the priming loop, including the conformational change of W800 that is suggested to be required for stabilizing the initiation complex. C. the MTase and polymerase domains shown in (A) are rendered as pink and red surfaces, respectively, to emphasize the expected large interface between the two domains. Residue T248 is colored black; substitution of this residue by an isoleucine could impact the interface between the two domains of NS5 structure. The figure was prepared using program CHIMERA.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005009.g002: Location of the mutation sites associated with altered fidelity in WNV NS5.A. Shown are the crystal structures of the N-terminal methyltransferase domain (MTase, colored pink; PDB 2OY0) and the C-terminal polymerase domain (RdRp, colored red; PDB 2HFZ) of the NS5 protein from WNV. The relative positioning of the two domains was based on the crystal structure of the full length NS5 protein of DENV (PDB 4V0R). The protein is represented as ribbon and the identified mutants (T248, V793, and G806) are represented as black spheres. The amino acids V793 and G806 are located in the priming loop that is part of the thumb subdomain. B. The initiation model of WNV RdRp showing the thumb subdomain of the polymerase, rotated 180° compared to the view in A, a 4-mer RNA extracted from the ɸ6-RdRp (PDB 1HI0, green carbon atoms), rNTP modeled at the priming site (P) and the catalytic site (C) based on the complex structure of HCV RdRp (PDB G1X5, yellow carbon atoms), and the active-site aspartates D636 and D669 with the bound catalytic Mg2+ ion. The substitutions, V793I and G806R, are expected to impact the interactions of the nearby residues of the priming loop, including W792, T799, W808, and M809. Disruption of these interactions would affect the dynamics of the priming loop, including the conformational change of W800 that is suggested to be required for stabilizing the initiation complex. C. the MTase and polymerase domains shown in (A) are rendered as pink and red surfaces, respectively, to emphasize the expected large interface between the two domains. Residue T248 is colored black; substitution of this residue by an isoleucine could impact the interface between the two domains of NS5 structure. The figure was prepared using program CHIMERA.
Mentions: In order to identify shared WNV amino acid (aa) substitutions associated with mutagen resistance, full-genome sequencing of clonal strains WNV pp3 and WNV pp9 was completed. A total of 15 (pp3) and 18 (pp9) nt substitutions were identified, resulting in 9 and 8 aa substitutions, respectively (Table 2). Of these, 10 nt and 7 aa substitutions were shared. Given the assumption that substitutions outside of the replication complex were more likely to be associated with adaptation to Hela cell culture or drift, shared aa substitution in the WNV RdRp and methyltransferase (Mtase) genes exclusively were chosen for further characterization. These included C8423T, resulting in a threonine to isoleucine change at position 248 of the Mtase, as well as G10057A and G10096A, resulting in valine to isoleucine and glycine to arginine changes at positions 793 and 806 of the RdRp, respectively (Table 2). Mapping of these residues on the known flavivirus RdRp and Mtase structures demonstrates that T248I is located at the C-terminal loop (aa 245–267), which is expected to interact with the RdRp domain, and both V793I and G806R exist in locations outside of the RdRp active site, although are within the priming loop (Fig 2; [39–42]). Both T248 and G806 are conserved among lineage I WNV strains, yet not across lineages or species, while V793 is shared among flaviviruses. No naturally circulating strains were found to possess the identified mutations at these locations.

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