Cholesterol is critical for Epstein-Barr virus latent membrane protein 2A trafficking and protein stability

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking signals induced by the B cell receptor (BCR) altering normal B cell development. LMP2A specifically associates with Nedd4 family ubiquitin-protein ligases which downmodulate LMP2A activity by ubiquitinating LMP2A and LMP2A-associated protein tyrosine kinases (PTKs). Since specific ubiquitin tags provide an endocytic sorting signal for plasma membrane proteins which traffic to membrane vesicles, we examined LMP2A localization and trafficking. We found that LMP2A is secreted through exosomes, small endocytic membrane vesicles, as previously demonstrated for LMP1. Interestingly, the treatment of cells with methyl-beta-cyclodextrin (MCD), which depletes cholesterol from plasma membrane, dramatically increased LMP2A abundance and LMP2A exosome secretion. Cholesterol depletion also blocked LMP2A endocytosis resulting in the accumulation of LMP2A on plasma membrane. LMP2A phosphorylation and ubiquitination were blocked by cholesterol depletion. LMP2A in the exosomal fraction was ubiquitinated but not phosphorylated. These results indicate that cholesterol-dependent LMP2A trafficking determines the fate of LMP2A degradation.