Additional file 1: Figure S1. of RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells

Summary of the process employed to select I. scapularis genes for RNAi knockdown experiments. Δ ISE6 proteins from the differential proteomic analysis at 36 hpi were analyzed. Proteins were selected based on (1) increased expression level, (2) strength of proteomic support (minimum 2 peptides identified from LC-MS-MS per protein) from proteins identified in Grabowski et al. [4], and (3) orthology to vertebrate/invertebrate proteins; * orthologous proteins identified in published proteomic studies [4–6, 8]. LGTV denotes proteins that exhibited increased expression following LGTV infection and LGTV & UV-LGTV denotes proteins that exhibited increased expression following both LGTV infection and UV-LGTV treatment. + proteins that exhibited increased expression following LGTV infection as compared to UV-LGTV treatment. FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, 1-pyrroline-5-carboxylate dehydrogenase; VNN, pantetheine hydrolase; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, UMP-CMP kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195, hypothetical protein; Hypo576. The prefix “ISCW” denotes VectorBase accession IDs. Figure S2 Effect of pGEM dsRNA concentrations on ISE6 cell viability following transfection for 60 h. X-tremeGENE (Xtr) transfection reagent was used to optimize pGEM dsRNA (RNAi negative control) concentrations in ISE6 cells at 60 h post transfection. Cell viability readings were compared to the Xtr + OptiMEM (Opti) control (gray bar). Red boxes indicate increased or no significant decrease in ISE6 cell viability. RLU560,590, relative light units 560 nm excitation and 590 nm emission. Error bars represent SEM. Statistical analysis was performed using an unpaired t-test between Xtr + Opti control and each pGEM dsRNA concentration. *p value ≤ 0.05 and **p value ≤ 0.01. Results represent 3 technical replicates and 1 biological replicate (multiple biological replicates completed with 10 ng concentration). Figure S3 Effect of transfection with dsRNA on ISE6 cell viability. FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, 1-pyrroline-5-carboxylate dehydrogenase; VNN, pantetheine hydrolase; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, UMP-CMP kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195, hypothetical protein; Hypo576, hypothetical protein; pGEM, pGEM plasmid (negative control; light gray bars); LGTV 3UTR, 3’ UTR of LGTV TP21 strain (positive control; dark gray bars), RLU560,590, relative light units 560 nm excitation and 590 nm emission. ISE6 cell viability following transfection with 10ng dsRNA for 60 h normalized to the negative control pGEM dsRNA. Results represent 2–5 technical replicates and 3 biological replicates. Error bars represent SEM and unpaired t-tests for comparison of cell viability of the negative pGEM control versus each gene of interest. Table S1 T7-tagged primers used to amplify cDNA and synthesize dsRNA. Table S2 Primers used to amplify cDNA for I. scapularis genes of interest by RT-qPCR. Table S3 Enrichment/cluster analysis of ISE6 proteins that exhibited increased expression following LGTV and UV-LGTV treatment. ISE6 proteins with increased expression following LGTV infection and/or UV-LGTV treatment from [4] were searched via DAVID enrichment analysis. For each cluster, the P value represents a modified Fisher Exact P value, and EASE score implemented in DAVID gene enrichment and functional annotation analysis. Enrichment (E) score of ≥ 1.3 is equal to P value of ≤ 0.05. Table S4 Nucleotide similarity of RT-PCR products amplified from I. scapularis and ISE6 cells and IscaW1 gene models. Table S5 Summary of statistically significant values corresponding to figures. (DOCX 329 kb

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

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

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

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