A novel dominant-negative mutant form of Epstein-Barr virus latent membrane protein-1 (LMP1) selectively and differentially impairs LMP1 and TNF signaling pathways.
The latent membrane protein-1 (LMP1) is an integral membrane molecule expressed by Epstein-Barr virus (EBV) during viral latency and displays properties of a constitutively activated member of the TNF receptor family. LMP1 is required for B-cell or monocyte immortalization induced by EBV and is sufficient to transform rodent fibroblasts. Transforming potential of LMP1 is mediated by its cytoplasmic C-terminal domain, which activates various cellular signaling pathways including NFkappaB and JNK. In this report, we constructed mutants of LMP1 with preserved membrane spanning domain but mutated in the C-terminal domain and a second truncated C-terminal LMP1 fused to the enhanced green fluorescent protein. This latter mutant, termed LMP1-CT, impairs signaling by ectopic LMP1 as well as endogenous EBV-expressed wild-type (wt) LMP1. In contrast to dominant-negative mutants of LMP1 with preserved membrane spanning domains, LMP1-CT was unable to bind wt LMP1 to form an inactive complex. Its dominant-negative effects were due to binding and sequestration of LMP1 adapters TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. The effect was selective since LMP1-CT did not inhibit IL-1beta-induced signaling, whereas it impaired TNF-triggered NFkappaB and JNK signals without affecting TNF-induced apoptosis. In addition and in contrast to LMP1 constructs with membrane localization, LMP-CT did not display cytostatic properties in noninfected cells. Importantly, LMP1-CT inhibited survival induced by LMP1 in an EBV-transformed T-cell line expressing the type II viral latency commonly found in the majority of EBV-associated human tumors. These data demonstrate that LMP1-CT is a new tool to explore the differences between LMP1 and TNF signaling and may facilitate the design of molecules with potential therapeutic roles.
Adriaenssens
Eric
E
CNRS UMR 8527, Institut de Biologie de Lille, BP 447, 59021 Lille Cedex, France.
Mougel
Alexandra
A
Goormachtigh
Gautier
G
Loing
Estelle
E
Fafeur
Véronique
V
Auriault
Claude
C
Coll
Jean
J
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005799:Genes, Dominant; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D008156:Luciferases / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D009000:Monocytes / Q000378:metabolism; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism / Q000821:virology; D047992:TNF Receptor-Associated Factor 2; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D016328:NF-kappa B; D011506:Proteins; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D014763:Viral Matrix Proteins; D018799:Intercellular Adhesion Molecule-1; D008156:Luciferases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases