Figure 3.

An E117A Mutation in LGTV NS3pro Disrupts TRAF6 Binding and Mature Protease Accumulation

(A) CoAP of mutant LGTV NS2B/3pro constructs with TRAF6. HEK293 cells were transfected with 2 μg of each indicated plasmid. Cell lysates were pulled down using streptavidin-conjugated beads at 24 h post-transfection, and eluted proteins were analyzed by immunoblot with indicated antibodies. Results representative of three independent experiments.

(B) Quantification of TRAF6 pull-down by mutant LGTV NS2B/3pro constructs in (A). Precipitated TRAF6-GFP from three independent experiments were quantitated using ImageJ after normalization to β-actin. Data are presented as percent pull-down of TRAF6-GFP relative to NS2B/3pro WT ± SD. ns, not significant; ***p < 0.001.

(C) Immunoblot analysis of whole-cell extracts from HEK293 cells expressing mutant LGTV NS2B/3pro constructs. HEK293 cells were transfected with 2 μg of each indicated plasmid and processed 24 h post-transfection. Blots were probed with indicated antibodies. Results representative of three independent experiments.

(D) Quantification of cleaved NS3pro of mutant LGTV NS2B/3pro constructs in (C). Cleaved NS3pro from three independent experiments were quantitated using ImageJ after normalization to β-actin. Data are presented as percent cleavage relative to NS2B/3pro WT ± SD. ns, not significant; ****p < 0.0001.

(E) The modeled structure of the complex of the LGTV NS3pro TBM2 (colored in magenta) with the C-terminal MATH domain of TRAF6 (colored in cyan). Dashed lines indicate dual hydrogen bonds between the omega oxygen atom of E117 of NS3 and the backbone amide N-H groups of L457 and A458 of TRAF6.

See also Figure S3.