FIGURE 4.

BICP0 binds TRAF6 though “346-PAERQY-351” and Y351 is the key residue. HEK293T cells (∼1 × 10⁷) were seeded in 100 mm dishes and transfected with the appropriate expression plasmids. At 36 h post-transfection, cell lysates were incubated with Anti-Flag/Anti-HA magnetic beads for 2 h at room temperature according to the manufacturer’s instructions. Western blot analysis with the indicated antibodies was performed. (A) Co-IP results by anti-Flag magnetic beads showed that BICP0 interacts with TRAF6 in the absence of other viral proteins. Co-IP (B) and reversed Co-IP (C) showed that the “346-PAERQY-351” motif of BICP0 responds for its interaction with TRAF6 and tyrosine 351 is the key amino acid. (D) Ubiquitination analyses revealed that the mutation of tyrosine 351 on BICP0 reduced TRAF6 K48-ubiquitination. Experiments were repeated at least three times and produced similar results.