Figure 2.

TRAF6 Is a Proviral Factor for TBFVs

(A) Representative images of confocal microscopy of LGTV-infected WT or TRAF6^−/− MEFs. MEFs were infected at an MOI of 1 and were processed at 24 hpi. Cells were immunostained with anti-E (green) and anti-NS3 (red) antibodies. Nuclei were stained with DAPI (blue); scale bar (white solid line), 200 μM.

(B) Titration of infectious particles in the supernatant from WT or TRAF6^−/− MEFs infected with LGTV (MOI of 0.1) for indicated times (h). Viral titers were determined by immunofocus assay (left y axis). IFN-β protein in supernatants from infected cells was quantitated by ELISA and is represented by a column graph (right y axis). Results are representative of three or more independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; *p < 0.05, ***p < 0.001.

(C) Immunoblot analysis of whole-cell extracts from WT or TRAF6^−/− MEFs infected with LGTV (MOI 0.1) for indicated times (h). Blots were probed with antibodies to LGTV NS3, LGTV E, IFIT2, IFIT3, TRAF6, and β-actin. Results representative of three or more independent experiments. See Figure S2A for longer exposures of LGTV NS3 and LGTV E.

(D) Titration of infectious particles in the supernatant from WT or TRAF6^−/− HEK293 cells expressing either GFP or TRAF6-GFP infected with LGTV (MOI of 0.1) at 24 hpi. Viral titers were determined by plaque assay. Whole-cell extracts from the infected cells were analyzed by immunoblot using GFP, TRAF6, and β-actin antibodies. Data are average of three independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; ****p < 0.0001.

(E–G) Titration of infectious particles in the supernatant from WT or TRAF6^−/− MEFs infected with TBEV (E), KFDV (F), or WNV (G) (MOI of 0.1) for indicated times (h). Viral titers were determined by plaque assay. Data are average of three independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

(H) Representative images of confocal microscopy of KUNV-infected WT or TRAF6^−/− MEFs. MEFs were infected at an MOI of 1 and were processed at 24 hpi. Cells were immunostained with anti-NS3 (green) antibodies. Nuclei were stained with DAPI (blue); scale bar (white solid line), 100 μM.

(I) Titration of infectious particles in the supernatant from WT or TRAF6^−/− MEFs infected with KUNV (MOI of 0.1) for indicated times (h). Viral titers were determined by plaque assay. Data are representative of three or more independent experiments performed in triplicate and plotted as mean ± SEM. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

(J) Immunoblot analysis of whole-cell extracts from WT or TRAF6^−/− MEFs infected with KUNV (MOI 0.1) for indicated times (h). Blots were probed with antibodies to WNV NS3, TRAF6, and β-actin. Results representative of three or more independent experiments.

See also Figure S2.