Figure 1.

LGTV NS3pro Interacts with TRAF6

(A) Sequence alignment of tick- or mosquito-borne flavivirus NS3pro. Putative TBMs are underlined, and anchor amino acids are highlighted in blue. Conserved residues of the catalytic triad are highlighted in red.

(B) Representative images of colocalization of LGTV NS3 and TRAF6. HEK293 cells expressing GFP or TRAF6-GFP were infected with LGTV at an MOI of 1. Cells were processed at 24 hpi, stained with anti-NS3 (red) and anti-GFP (green) antibodies, and visualized by confocal microscopy. Nuclei were stained with DAPI (blue); scale bar (white solid line), 10 μM. Yellow represents co-localized proteins. White box indicates area of enlargement in inset panel; inset scale bar (white solid line), 5 μM. White arrowhead indicates a distinct TRAF6 punctum observed in uninfected cells.

(C) CoIP of LGTV NS3 and endogenous TRAF6 during infection. HEK293 cells were infected with LGTV at an MOI of 1. Cell lysates were prepared at 48 hpi, incubated with either anti-NS3 or control IgY antisera, and precipitated with PrecipHen beads. Samples were analyzed by immunoblot with the indicated antibodies. Results representative of two independent experiments.

(D) CoIP of LGTV NS3 and TRAF6. HEK293 cells expressing TRAF6-GFP were infected with LGTV or KUNV at an MOI of 10. Cell lysates were immunoprecipitated with anti-GFP antibody and protein-A-conjugated agarose beads at 48 hpi. Samples were analyzed by immunoblot with the indicated antibodies. Results representative of three independent experiments.

(E) CoAP of recombinant LGTV NS3 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 or more independent experiments. POI, protein of interest.

See also Figure S1.