Survivorship of AMCRs inoculated with WN/IC NS3-249 mutants.

<p>(A) Survivorship of Colorado AMCRs (n = 8) inoculated with WN/IC clone-derived viruses demonstrating variable amino acids at the NS3-249 locus (249P, 249D, 249T, 249H, and 249A); (B) Survivorship of AMCRs collected in 2012 (n = 4) inoculated with WN/IC clone-derived viruses (NS3-249P and 249N).</p

Viremia profiles of AMCRs inoculated with WN/IC NS3-249 mutants.

<p>(A) Mean daily viremias from AMCRs (n = 8) from Colorado inoculated with WN/IC NS3-249 point mutants (NS3-249P, 249D, 249T, 249H, and 249A); (B) Mean daily viremias from 2012 captured AMCRs (n = 4) inoculated with WN/IC NS3-249P and 249N mutants. Bars denote standard deviations from the mean.</p

Viremia profiles of HOSPs inoculated with WN/IC NS3-249 mutants.

<p>(A) Mean daily viremias from HOSPs (n = 8) from California inoculated with WN/IC NS3-249 point mutants (NS3-249P, 249D, 249T, 249H, and 249A); (B) Mean daily viremias from HOSPs (n = 4) from Colorado inoculated with WN/IC NS3-249P and 249N mutants. Bars denote standard deviations from the mean.</p

Temperature sensitivity assessment in an avian (DEF) cell line performed at 37°C (A) and 44°C (B).

<p>Cells were inoculated at an MOI of 0.1 and titers determined by plaque titration on Vero cells. (C) Depicts a compilation of the differential growth by subtracting titers determined from growth at 37°C from those observed at 44°C.</p

Sequence alignment of NS3-235–282 and helicase structure.

<p>(A) Alignment of 36 WNV strains between NS3 aa positions 235–282. The NS3-249 locus is indicated and the amino acid identities colored according to the same color scheme as depicted in panels A and B. Shaded areas correspond to aa 235–243 and aa 256–282, and emboldened and underlined text highlight genetic differences as well as sites of compensatory mutations (NS3-244 and NS3-259). Panel (B) Surface image depiction of the WNV helicase. Arrows depict RNA-entry site, ATP hydrolysis site and NS3-249 (yellow). Other substitutions identified in this study (salmon) include Q244H just above Pro249 and D259E just behind Q244. The peptides surrounding Pro249 include amino acids 256–282 (light cyan), 243–254 (dark cyan) and 235–243 (medium cyan).</p

Phenotypic and genetic characterization of rescued WN/IC NS3-249 mutants.

<p>(A) WN/IC NS3-249 point mutant plaque phenotypes in a mammalian (Vero) cell line. Plaque diameters were determined to be 1.8±0.3 mm (249P), 1.7±0.4 mm (249T), 1.4±0.3 mm (249A), 1.6±0.3 mm (249H), 1.3±0.2 mm (249D) and 1.9±0.5 mm (249N; not shown). (B) Chromatogram depicting the sequence identity of the NS3-249 loci (genomic position 5456-5458) following generation of the recombinant viruses. (C) WN/IC NS3-249 point mutant growth profiles in a mammalian (Vero) cell line. Cells were inoculated at an MOI of 0.1. Bars represent standard deviation from the mean.</p

NS3-249 point mutant ATPase kinetics during dsRNA unwinding.

<p>(A) ATPase activity with different concentration of NS3 protein. Lane 1: no protein, with 62.5 nM of recombinant protein (lane 2, 5, 8, 11 and 14), 125 nM of protein (3, 6, 9, 12 and 15), and 250 nM of protein (lane 4, 7, 10, 13 and 16). (B) Percent ATP hydrolyzed by each NS3-249 helicase protein at concentrations of 125 nM.</p

Phylogenetic analyses of genomic WNV strains.

<p>(A) Phylogenetic tree of the coding region of 36 WNV strains, constructed using Bayesian analysis (B) Maximum likelihood phylogenetic tree. Isolates are colored according to the amino acid at position 249 in NS3. Magenta  =  Pro; Blue  =  Thr; Green  =  His; Orange  =  Ala; Black  =  Asn. Asterisks represent nodes supported by at least 95% posterior probability. Clades are highlighted by WNV lineage. Pink  =  Lineage 1a; Orange  =  Lineage 1b; Yellow  =  Lineage 1c; Green  =  Lineage 2; Gray  =  Lineage 3; Blue  =  Lineage 4.</p

Helicase activity of recombinant WNV helicase NS3-249 protein mutants.

<p>(A) Helicase activity with fixed concentration of NS3 (125 nM). Lane 1: ssRNA (heat denature dsRNA), Lane 2: dsRNA (no protein), Lane 3: NS3-249P, Lane 4: NS3-249A, Lane 5: NS3-249D, Lane 6: NS3-249H, Lane 7: NS3-249T. (B) Percent dsRNA unwound by each NS3-249 helicase point mutants at 125 nM. (C) Helicase activity with increasing concentrations of NS3 (125 nM, 250 nM, 500 nM and 1,000 nM).</p