Fig 5.

Effects of expression of Rec and Staufen-1 on HERV-K(HML-2) virus particle production. (A) HEK 293T cells were cotransfected with Rec-V5, Staufen-1, and oriHERV-K113, as indicated. Supernatants were filtered and pelleted by ultracentrifugation, and virus particles were detected by a HERV-K(HML-2) anticapsid polyclonal antibody. (B) Downregulation of Staufen-1 expression by a mix of Staufen-1-specific shRNAs. The top panel shows results of Western blotting with lysates of transfected HEK 293T cells. The blot was subsequently probed with an antitubulin antibody as a loading control. (C) Demonstration of the shRNA effect at the single-cell level. Cells transfected with pLVTHM_shRNAs (indicated by white arrows) show almost no staining with rat anti-Staufen-1 serum, while cells transfected with the empty vector pLVTHM (marked by stars) show staining similar to that of untransfected cells. The pLVTHM vector expresses GFP as a control. (D) In contrast to cells transfected with the empty pLVTHM vector, transfection with pLVTHM_shRNA significantly reduces the production of HERV-K113 virions. The experiment was repeated twice with duplicates. The means and standard errors are shown.