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Open Access Research

Characterization of the latent membrane protein 1 signaling complex of Epstein-Barr virus in the membrane of mammalian cells with bimolecular fluorescence complementation

Pooja Talaty, Amanda Emery and David N Everly*

Author Affiliations

Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, Illinois, 60064, USA

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Virology Journal 2011, 8:414  doi:10.1186/1743-422X-8-414

Published: 24 August 2011

Abstract

Background

Bimolecular fluorescence complementation (BiFC) is a novel technique to examine protein-protein interaction through the assembly of fluorescent proteins. In the present study, BiFC was used to study the assembly of the Epstein-Barr virus latent membrane protein 1 (LMP1) signaling complex within the membrane of mammalian cells. LMP1 signaling requires oligomerization, localization to lipid rafts, and association of the cytoplasmic domain to adaptor proteins, such as the tumor necrosis factor receptor associated factors (TRAFs).

Methods

LMP1-TRAF and LMP1-LMP1 interactions were assayed by BiFC using fluorescence microscopy and flow cytometry. Function of LMP1 BiFC contructs were confirmed by transformation assays and nuclear factor- κB (NF-κB) reporter assays.

Results

BiFC was observed between LMP1 and TRAF2 or TRAF3 and mutation of the LMP1 signaling domains reduced complementation. Fluorescence was observed in previously described LMP1 signaling locations. Oligomerization of LMP1 with itself induced complementation and BiFC. LMP1-BiFC constructs were fully functional in rodent fibroblast transformation assays and activation of NF-κB reporter activity. The BiFC domain partially suppressed some LMP1 mutant phenotypes.

Conclusions

Together these data suggest that BiFC is a unique and novel platform to identify and characterize proteins recruited to the LMP1-signaling complex.

Keywords:
Epstein-Barr Virus; latent membrane protein 1; bimolecular fluorescence complementation; TRAF2; TRAF3