High Physiological Levels of LMP1 Result in Phosphorylation of eIF2α in Epstein-Barr Virus-Infected Cells

Abstract

LMP1 is an Epstein-Barr virus (EBV)-Latent membrane protein 1 (LMP1), an Epstein-Barr virus (EBV)-encoded oncoprotein, mimics an active cellular receptor, CD40, but in a ligand-independent manner (15, 36). LMP1 stimulates multiple signaling pathways, including NF-B-, JNK-, and STAT-mediated transcription (10,14,29). As an oncoprotein, LMP1 is essential for maintaining the proliferation of EBV-infected lymphoblasts (9, 26), it transforms rodent fibroblasts to proliferate anchorage independently (1, 38), and it also induces B-cell lymphomas in transgenic mice late in their lives (28). LMP1 is an integral membrane protein consisting of a short amino cytoplasmic domain, a six-membranespanning domain, and a 200-amino-acid carboxy cytoplasmic domain. The carboxy cytoplasmic domain binds TRAFs, TRADD, and JAK3 and is essential for LMP1's ability to maintain proliferation of EBV-positive lymphoblasts and to activate multiple signaling pathways (9,10,14,20,29,30,33).In contrast to its ability to stimulate signaling and cell proliferation positively, LMP1 also inhibits RNA accumulation and general protein synthesis and induces cytostasis when it is expressed at levels as little as twofold higher than the average for EBV-infected cells (12,24,34). This inhibition requires the first two membrane-spanning domains of LMP1, but the mechanism of inhibition is unknown (5). Because all of the studies of this inhibitory activity have been carried out in transfected cells, it was unclear whether the inhibition occurs in EBVinfected cells and thus whether it could affect the EBV life cycle.We have sought to understand this inhibitory function of LMP1 by first determining if it occurs in EBV-infected cells. We have found that in three different clones of EBV-infected lymphoblasts, the levels of expression of LMP1 in individual cells in each clone range over 100-fold. This variation in LMP1 protein levels correlated with LMP1 RNA levels. Cell proliferation was diminished in individual cells expressing high levels of LMP1, which is consistent with the notion that high natural levels of LMP1 inhibit cell proliferation and indicates that the inhibitory effect of LMP1 occurs under physiological conditions. We next investigated the mechanism by which LMP1 inhibits gene expression. We found that higher levels of LMP1 result in the phosphorylation of eukaryotic translation initiation factor 2 (eIF2␣) that is dependent upon LMP1's transmembrane domains but not its carboxy-terminal signaling domain. Importantly, this induction of phosphorylation of eIF2␣ occurs normally in EBV-infected cells. In EBV-infected clones, cells expressing high physiological levels of LMP1 had high levels of phosphorylation of eIF2␣. Our results indicate that LMP1 inhibits gene expression and cell proliferation when expressed at high physiological levels. This inhibition occurs as one extreme of the normal spectrum of activities mediated by LMP1. MATERIALS AND METHODSCell culture and plasmids. 721 is an EBV-positive lymphoblastoid cell line (23). 11/17-2 and 11/17-3 ...

Epstein–Barr virus encoded LMP1 downregulates TCL1 oncogene through miR-29b

“…The reasons for LMP1 correlated reduced cellular proliferation might be several fold. It might be due to regulation of pro-apoptotic genes induced by LMP1 (Dirmeier et al, 2005) or reduction of gene expression through phosphorylation of elongation factor (Sandberg et al, 2000;Lam et al, 2004). The present data that several tumor suppressor miRs are induced by LMP1 and the downregulation of TCL1 through miR-29b adds another mechanistic clue as to cytostasis provoked by this protein in B lymphoma cells.…”

The latent membrane protein 1 oncogene modifies B-cell physiology by regulating autophagy

“…Those with intermediate levels of LMP1 will transit it most efficiently and thus constitute the majority of the population. Those with high levels of LMP1 are inhibited in their general protein synthesis and can be arrested throughout the cell cycle until the levels of LMP1 decrease (Lam et al, 2004). EBV has evolved a mechanism that drives cells with extremes in levels of LMP1 to evolve to express it at intermediate levels and to proliferate to allow the population's continued exponential growth.…”

“…BJAB/HALMP1 cells (Lam et al, 2004) were treated with tetracycline to induce the expression of LMP1. Expression of LMP1 elicited the processing of endogenous LC3, which is a distinctive feature of induction of autophagy ( Figure 1a).…”

“…To determine if the expression of the six transmembrane domains of LMP1 alone can induce autophagy, BJAB/HA6MLMP1-green fluorescent protein (GFP) cells were used. This cell line has been engineered to express a derivative of LMP1 that expresses only the N terminus and six transmembrane domains of LMP1 conditionally upon treatment with tetracycline (Lam et al, 2004). Western blot analysis showed that the level of LC3II was increased in cells expressing the six transmembrane domains of LMP1 (Figure 1b).…”

“…This moiety regulates LMP1's trafficking, aggregation and dose-dependent induction of the unfolded protein response (UPR) Kaykas et al, 2002;Lee and Sugden, 2007, submitted). Second, LMP1 is expressed at levels varying over 100-fold in individual cells of clonal populations (Lam et al, 2004). This variation is controlled in part autocatalytically and requires LMP1's induction of the UPR (Lee and Sugden, 2007, submitted).…”