A novel dominant-negative mutant form of Epstein–Barr virus latent membrane protein-1 (LMP1) selectively and differentially impairs LMP1 and TNF signaling pathways

Abstract

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 NFjB 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 EBVexpressed wild-type (wt) LMP1. In contrast to dominantnegative 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 IL1b-induced signaling, whereas it impaired TNF-triggered NFjB 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 EBVassociated 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.

Autoactivation of the Epstein-Barr Virus Oncogenic Protein LMP1 during Type II Latency through Opposite Roles of the NF-κB and JNK Signaling Pathways

“…pSVHA, pSVHA-LMP1, pSVHA-LMP1-TM, and LMP1-CT constructs were all previously described (2). pSVHA-LMP1-Tes1mut and pSVHA-LMP1-Tes2mut mutants were generated by site-directed mutagenesis; codons 204 to 208 in TES1 were mutated from PXQXT to AXAXA, and the codons 384 to 386 YYD in TES2 were deleted.…”

“…We have described and extensively characterized two cell lines (TE1 and NC5) in which EBV was found to express a type II latency (20,39,40). Moreover, as already shown by others studying LCLs (27), we have demonstrated by two different approaches (i.e., antisense oligonucleotides and our original dominant-negative mutant, LMP1-CT) that LMP1 is essential for proliferation and survival of both of our EBV-transformed models of type II latency (2,39). Owing to this essential role in EBV-dependent oncogenesis and since it can transform rodent fibroblasts (61) and sensitizes transgenic mice to lymphomas (31), LMP1 is considered the main EBV oncogene.…”

Regulation of DNA Polymerase β by the LMP1 Oncoprotein of EBV through the Nuclear Factor-κB Pathway

“…3B). The cleavage product of LMP1, due to an endogenous cleavage site (24), was also observed (Fig. 3B).…”

“…2B). We then repeated the experiment with both cell lines stably transfected with a doxycyclin inducible vector coding for the LMP1CT artificial variant (22), which has a dominant-negative effect on LMP1 intracellular signaling (24). Expression of LMP1CT in estrogen-starved EREB2-5 cells failed to up-regulate Polβ (Fig.…”

“…The cDNAs coding for IκBα S32,36A (constitutively active form of IκBα; ref. 23), LMP1CT (a dominant-negative mutant of LMP1) and LMP1-B95.8 were cloned (24).…”