FIG 4.

The enhancement of type I IFN induction by F1a is correlated with the proteasome targeting activity. (A) Proteasome activity disturbance level by the ectopic F protein expressions was analyzed by flow cytometry for the ZsGreen-positive cells. F1a-, F1a126-, and F1a/2a-expressing cells had higher ZsGreen-positive cells than those in the control and F2a-expressing cells. (B) Similar to panel A, the experiment was repeated in the presence of MG132, and F1a-, F1a126-, and F1a/2a-expressing cells showed the same phenotype with higher ZsGreen-positive cells present than those in the control and F2a-expressing cells. (C) The bar graph shows the summarized flow cytometry results of panels A and B (n = 3). Means with the same letter are not significantly different from each other by t test (P < 0.05). (D) Ectopic expression of F proteins in Huh7 cells containing the pZsProSensor-1 plasmid. Huh7 cells containing pZsProSensor-1 plasmid cells were transfected with different constructs of F proteins and harvested at 48 h posttransfection for anti-HA immunoblotting. Cells were treated with MG132 for 6 h before harvesting. (E) Flag-tagged PSMA3 and HA-tagged F proteins were cotransfected into Huh7 cells. Proteins were recovered by anti-Flag co-IP and identified by anti-HA or anti-Flag immunoblot analysis. (F and G) Flag-tagged PSMA3 (F) or Flag-tagged PSMA7 (G) and HA-tagged F proteins were cotransfected into HEK293 cells. Proteins were recovered by anti-Flag co-IP and identified by anti-HA or anti-Flag immunoblot analysis. IP, immunoprecipitation.