FIG. 4.
Identification of critical residues within LGTV NS5:355-735 required for JAK-STAT inhibition. (A) Summary of site-directed mutations made in NS5:342-735. The number of each amino acid within the RdRP is indicated in blue (finger subdomain), red (palm subdomain), or green (thumb subdomain). The eight conserved RdRP motifs within LGTV NS5 are boxed (I through VIII). Amino acids represented in yellow indicate site-directed substitutions to Ala that resulted in a WT phenotype of pY-STAT1 inhibition by IFA. Those marked in red indicate site-directed substitutions to Ala that resulted in a mutant phenotype of pY-STAT1 inhibition by IFA. An asterisk indicates a strong change in phenotype, with approximately 80% of V5-positive cells containing nuclear pY-STAT1. Red boxes without an underlying asterisk indicate a moderate change in phenotype, with approximately 20 to 50% V5 positive cells also containing detectable pY-STAT1. (B) IFA of select site-directed mutants of LGTV NS5:342-735. Vero cells expressing each construct were treated with IFN-β and stained for the V5 epitope tag (red) and phosphorylated STAT1 (pY-STAT1; green). The DD664/665AA mutant exhibited a WT phenotype of inhibition, whereas all others depicted here had a mutant phenotype. (C) Quantification of pY-STAT1 inhibition by each full-length NS5 construct analyzed by flow cytometry. Error bars indicate SEM; asterisks indicate significant differences from WT LGTV NS5 (P < 0.05 by ANOVA followed by Tukey's test).