Epstein-Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase

Epstein-Barr virus (EBV) stimulates the proliferation of latently infected B cells and promotes lymphoid malignancies in humans. To address the role of EBV latency protein Epstein-Barr nuclear antigen 3C (EBNA3C) in regulation of the retinoblastoma protein (Rb), we transfected EBNA3C into 293, BJAB, and SAOS-2 cells. In this context, a dominant effect of EBNA3C is to decrease Rb protein levels. EBNA3C also rescues an Rb-induced flat cell phenotype and targets Rb for proteasome- and ubiquitin-dependent degradation. Further, EBNA3C forms a stable complex with Rb in cells when the proteasome machinery is inhibited and interacts with Rb in vitro, mapping to a conserved domain at the terminus of EBNA3C. Deletion analysis of EBNA3C identified a motif within amino acids 140-149 important for both the binding and regulation of Rb. This motif is of particular interest, because it has also been linked to regulation of the Skp1/Cul1/F-box complex, SCF^Skp2. Indeed, inhibition of Skp2 function with a dominant-negative molecule reduces the ability of EBNA3C to degrade Rb. Skp2 has no detectable effect on Rb levels in the absence of EBNA3C, suggesting that SCF^Skp2 is specifically usurped by EBNA3C for the enhancement of Rb degradation. That EBNA3C has exploited this association suggests that other human malignancies might use a similar strategy to regulate the Rb protein.