Functional analysis of the mutated Epstein-Barr virus oncoprotein LMP1(69del): implications for a new role of naturally occurring LMP1 variants

Abstract

BACKGROUND AND OBJECTIVES: The role of carboxyterminal deletions of the latent membrane protein-1 (LMP1) in Epstein-Barr virus (EBV) infection and oncogenesis is unclear. Here we describe functional properties of a rare 69-bp LMP1 deletion mutant (LMP1(69del)) isolated from a patient with polyclonal B-cell lymphocytosis. DESIGN AND METHODS: Colony focus assay was used to evaluate the transforming capacity of LMP1(69del) in comparison to that of wild-type LMP1 from EBV strain B95/8. Transient transfectants of B-, T-, epithelial and 3T3 cells, and stable transfectants with ecdysone-inducible LMP1 expression were produced. The signaling capacity of both LMP1s on nuclear transcription factors NFkappaB and AP-1 were studied. Secretion of matrix metalloproteinase MMP-9, apoptosis, and EBV lytic and latent gene expression were also investigated. RESULTS: LMP(69del) showed transforming properties comparable to those of the wild-type oncoprotein. Induction of NFkappaB but a markedly reduced influence on AP-1 were observed. Both oncoproteins induced secretion of MMP-9, and enhanced pre-apoptotic effects in Jurkat-T cells leading to increased Fas/Apo-1 and doxorubicin-mediated apoptosis. Furthermore, LMP1(69del) showed a more effective down-regulation of the EBV lytic cycle master gene BZLF1(Zebra) than did wild-type LMP1. INTERPRETATION AND CONCLUSIONS: (i) LMP1(69del) possesses oncogenic properties, (ii) the observed impaired activity on AP-1 does not interfere with MMP-9 induction, (iii) the enhanced inhibition of BZLF1 could compensate for previously described mutations of our isolate leading to a more lytic phenotype and may be responsible for counteracting permanent virus replication in the chronic active EBV syndrome observed in this patient.