FIG 7.

EEF1A1 stabilizes components of the viral replicase complex. (A) EEF1A1 enhances JEV replication by increasing the interaction amount between it and viral replicase components. HEK293T cells were transfected with miR-33a-5p mimics or control mimics and, 24 h later, the cells were infected with JEV at an MOI of 1.0. Cell lysates were harvested at 36 h postinfection, and protein concentrations were measured and quantified. The cell lysates were then subjected to coimmunoprecipitation with anti-NS5 antibodies. The precipitates were analyzed by Western blotting with anti-NS3, anti-NS5, or anti-EEF1A1 antibodies. (B) HEK293T cells containing control shRNA or EEF1A1 shRNA, or HEK293T cells containing miR-33a-5p mimics or control mimics, were infected with JEV at an MOI of 1.0. Cell lysates were harvested at 36 h posttransfection and immunoprecipitated with anti-NS3 or anti-NS5 antibodies. The precipitates were subjected to Western blotting with anti-NS3, anti-NS5, and anti-K48-ubiquitin antibodies. (C) HEK293T cells containing control shRNA or EEF1A1 shRNA, or HEK293T cells containing miR-33a-5p mimics or control mimics were infected with JEV at an MOI of 1.0. In one set of experiments, 20 nM MG132 was added at 24 h postinfection to inhibit proteasomal degradation. Cell lysates were prepared at 12 h after treatment and analyzed by Western blotting with anti-NS3, anti-NS5, and anti-EEF1A1 antibodies.