Epstein-Barr Virus Latent Membrane Protein 1 (LMP-1) Half-Life in Epithelial Cells Is Down-Regulated by Lytic LMP-1

Abstract

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is associated with numerous malignancies, especially nasopharyngeal carcinoma (NPC). Oncogenic EBV latent membrane protein 1 (LMP-1) is a 63-kDa protein of 386 amino acids (strain B958) encoded by the BNLF1 gene ( Fig. 1). LMP-1 inserts into the plasma membrane and functions as a ligand-independent, constitutively active growth factor receptor, similar to CD40 of the tumor necrosis factor receptor (TNFR) family. After oligomerization, LMP-1 binds TNFRassociated factors (TRAFs) and the TNFR-associated death domain protein (26, 38) to activate intracellular signaling through the NF-B, cJun N-terminal kinase, and p38 mitogenactivated protein kinase pathways (10,11,42). LMP-1 also activates the JAK-STAT signaling pathway, possibly through interaction with Janus kinase 3 (JAK3) (19,22). Cumulatively, these signals generate many effects on host cell growth, differentiation, apoptosis, and immune response.The LMP-1 gene manifests remarkable natural sequence heterogeneity (9,35,48). Although a specific LMP-1 genotypedisease phenotype relationship has not yet been identified (48), NPC-derived LMP-1 is both structurally and functionally different from the LMP-1 derived from the laboratory EBV strain B958 in the following ways: (i) NPC LMP-1 activates NF-B signaling and AP-1 transactivation better than B958 LMP-1 (5,7,15,16,27,33,34), (ii) NPC LMP-1 down-regulates cell immune markers, blocks cell apoptosis, and up-regulates the epidermal growth factor receptor (EGFR) better than B958 LMP-1 (7,8,15,27,33,34,52), and (iii) NPC LMP-1 transforms cells and forms tumors in mice more efficiently than B958 LMP-1 (8,24,28,33,52). Chimeric studies of LMP-1 have mapped some of these functional differences to the carboxy-terminal 30-nucleotide domain encoding amino acids 343 to 352 (28) and transmembrane domain amino acids 85 to 129 (5, 34).The intracellular quantity of expressed LMP-1 and the cell type background together influence the strength of LMP-1 signaling and its ultimate phenotypic effect on the cell (4,16,17,20,24,28,49,50). The intracellular half-life of LMP-1 is g...

Oncogenic Activity of Epstein-Barr Virus Latent Membrane Protein 1 (LMP-1) Is Down-Regulated by Lytic LMP-1

“…The results of this study also lend strong support to the hypothesis that lyLMP-1 exerts its down-regulatory effect on LMP-1 by promoting LMP-1 degradation and thereby reducing the quantity of LMP-1 available to generate oncogenic signals. In this study, we demonstrated a dose-dependent reduction in LMP-1 oncogenic activity by lyLMP-1 that mirrors the previously demonstrated dose-dependent reduction in LMP-1 half-life by lyLMP-1 (24). More importantly, we demonstrated a reduction in expressed LMP-1 quantity by lyLMP-1.…”

“…Taken together, these results indicate that clone K confers a greater growth and survival advantage to epithelial cells than does clone B958WT at all three serum concentrations and that lyLMP-1 completely reverses the growth and survival advantage of clone K. tein quantities are determined not only by the quantity of each plasmid transfected and its relative transfection efficiency but also by the relative rates of synthesis and degradation for each protein. It is especially difficult to determine the stoichiometry of a protein-protein interaction when one of the coexpressed proteins acts to promote the degradation of the other, as lyLMP-1 does to LMP-1 (24) and as LMP-1 may do to lyLMP-1 (Fig. 5).…”

“…Unlike LMP-1, lyLMP-1 neither activates signaling nor transforms cells, and lyLMP-1 may even negatively regulate some LMP-1 signaling activity (7,31). Recently, we demonstrated that expression of lyLMP-1 significantly down-regulates the half-life of LMP-1 in a dose-dependent manner in epithelial cells (24). In this study, we tested the hypothesis that lyLMP-1 possesses the ability to down-regulate a broad range of LMP-1 signaling activities and the associated oncogenic cell phenotype induced by LMP-1 signaling.…”

Sequence variations of Epstein–Barr virus LMP1 gene in nasal NK/T-cell lymphoma

“…Antigenic analysis indicated that this variant sequence (YFLEILWRL) was not only poorly recognized by epitope-specific CTL but also showed significant loss in HLA A2 binding compared to the B95-8 sequence [25]. Pandya and Walling [48] showed that the amino acid change at codon 129 (Met to Ile) increased the LMP1 half-life in epithelial cells. It is likely that the variant sequence at codon 125-133 (YFLEILWRL) may be selected in nasal NK/T-cell lymphoma as well as NPC and may play important roles in reduced recognition of CTL and/or in lymphomagenesis.…”

A new model of LMP1–MYC interaction in B cell lymphoma

“…[28]). Wild-type LMP1 constitutively signals when assembled at cellular membranes and has a half-life of 4–8 h [29]. Using our inducible model of mCD40–LMP1-induced signaling, we show LMP1-mediated expression of both isoforms of Pim1 after 2 h, well within the surface membrane half-life of wild-type LMP1, and with similar kinetics to CD40 induction of Pim1 expression (2 h) [30].…”