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

Abstract

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.

Figure 1. LMP2A exosomal secretion

(A) LMP2A-mutated (ΔLMP2A) or LMP2A-expressing (WT) LCL and (B) LMP2A-expressing BJAB cell lines, C12 and F1, and control cell lines, C1 and A4, were grown for six days without changing the media. Cell culture media was removed from 0–6 days and cells were pelleted at 1,500 rpm (cellular fraction). The resulting supernatant was cleared by centrifugation at 3,000 rpm and exosomes were isolated by centrifugation at 30,000 rpm (exosomal fraction). Pelleted cell and exosomal fractions were solubilized and immmunoblotted with LMP2A, HLA-DR, and LAMP1. The reduction in HLA-DR expression in the late time points is likely due to MCD toxicity.

Figure 2. The effect of various chemicals on the abundance of LMP2A in the cellular and exosomal fractions

WT LCL was treated with indicated chemicals for 3 days. Cell and exosome fractions were immunoblotted with LMP2A and HLA-DR. Lactacystin (LC), Chloroquine (CQ), Methyl-beta-cyclodextrin (MCD), Sodium vanadate (Van), Genistein (Gen), and Src inhibitor PP2.

Figure 3. MCD specifically increases LMP2A in the cellular fraction and the exosomal fraction

(A) WT LCL was treated with 4 mM MCD for indicated times. Cellular and exosomal fractions were immunoblotted for LMP2A, LMP1 and HLA-DR. (B) WT LCL was treated with 4 mM MCD or 4 mM MCD plus 30 μg/ml soluble choresterol (Cho) for 24 hours. Cellular and exosomal fractions were immunoblotted with LMP2A.

Figure 4. Increased abundance of LMP2A by MCD is due to the protein stability and not de novo protein synthesis

(A) WT LCL was treated with 4 mM MCD in the presence of 100 μg/ml cycloheximide (CHX). Cell lystates were isolated at indicated times and immunoblotted with LMP2A and GAPDH. (B) The amount of LMP2A was densitometrically determined and plotted as a relative ratio. One of three comparable experiments is shown.

Figure 5. MCD translocates LMP2A to the plasma membrane

WT LCL was treated with 4 mM MCD for six hours and fixed with methanol. Localization of LMP2A and HLA-DR was visualized by reacting with anti-LMP2A or anti-HLA-DR, and followed by staining with FITC-conjugated goat anti-rat-immunoglobulin antibody or Cy3-conjugated goat anti-mouse-immunoglobulin antibody, respectively.

Figure 6. Phosphorylation and ubiquitination of MCD-treated LMP2A

WT LCL was treated with 4 mM MCD for 48 hours. Cellular and exosomal fractions were immunoprecipitated with rabbit anti-LMP2A, and immunoblotted with LMP2A, phosphotyrosine and ubiquitin. One of three comparable experiments is shown. * indicates cell-derived immunoglobulin heavy chain.

Figure 7. Model for LMP2A trafficking

(1) LMP2A is synthesized in the endoplasmic reticulumn and transported to the trans-Golgi network (TGN) prior to being transported to the plasma membrane. LMP2A is not phosphorylated and ubiquitinated (P⁻U⁻). (2) Once in the plasma membrane, LMP2A localizes in the lipid rafts forming multi-protein complexes with the Lyn and Syk PTKs and the Nedd4-family ubiquitin ligases resulting in the phosphorylation and ubiquitination of LMP2A (P⁺U⁺). (3) The LMP2A recruitment into the lipid rafts and subsequent phosphorylation and ubiquitination results in internalization and trafficking of LMP2A to early endosomes (EE). The MCD-mediated increase in LMP2A in cellular fractions is likely explained by LMP2A accumulation in the plasma membrane due to a block in LMP2A internalization. (4) Some LMP2A is recycled back to the plasma membrane through recycling endosomes. (5) LMP2A is further transported to multivesicular bodies (MVB)/late endosomes (LE) where LMP2A-containing membrane is invaginated into the lumen of MVB/LE. During the transportation from EE to MVB/LE, LMP2A dephosphorylation occurrs. However, whether LMP2A dephosphorylation induces this transportation to MVB/LE or LMP2A is dephosphorylated as it is transported, is still unclear. (6) LMP2A destined to lysosomes is degraded. (7) Some LMP2A is released to extracellular domains by fusing to the plasma membrane. In the exosome fractions, LMP2A is highly ubiquitinated but not phosphorylated (P⁻U⁺). When LMP2A endocytosis is blocked at the plasma membrane by MCD, increases in LMP2A exosomal secretion may be mediated by a poorly described mechanism in common for other proteins as described in the discussion and/or by the transport of LMP2A directly from TGN to MVB/LE (8).