Figure 2

Effects of UL56 and other viral proteins with a PY motif on Itch. (A) Itch is markedly decreased in cells stably expressing UL56 (Vero-GFP-UL56). Lysates from Vero, Vero-GFP-UL56, or Vero-GFP cells were analyzed for Itch and other Nedd4-family ubiquitin ligases. Itch was markedly decreased in Vero-GFP-UL56 cells. (B) VP5 did not decrease endogenous Itch. Vero cells were transfected with plasmids encoding VP5 (pcDNA-VP5) or control plasmids (pcDNA3.1(+)). The levels of Itch did not change in cells transfected with pcDNA-VP5. (C) VP5 and VP16, but not ICP0, caused the decrease of exogenous Itch. Vero cells were co-transfected with plasmids encoding Itch (pcDNA-Itch) and plasmids encoding a viral protein (pMyc-ICP0, pcDNA-VP5, or pcDNA-VP16) or control plasmids (pCMV-Myc or pcDNA3.1(+)). The levels of Itch decreased in cells transfected with pcDNA-VP5 or pcDNA-VP16. (D) Overexpression of Itch has no effect on the protein levels of VP5, VP16, or ICP0. Vero cells were co-transfected with plasmids encoding a viral protein (pMyc-ICP0, pcDNA-VP5 or pcDNA-VP16) and either pcDNA-Itch or control plasmids (pcDNA-3.1(+)). The levels of viral proteins did not change with the overexpression of Itch. β-actin was used as a loading control.