Identification of Transmembrane Protein 134 as a Novel LMP1-Binding Protein by Using Bimolecular Fluorescence Complementation and an Enhanced Retroviral Mutagen

Abstract

Latent membrane protein 1 (LMP1) of Epstein-Barr virus induces constitutive signaling in infected cells. LMP1 signaling requires oligomerization of LMP1 via its transmembrane domain, localization to lipid rafts in the membrane, and association of the LMP1 cytoplasmic domain to adaptor proteins, such as the tumor necrosis factor receptor-associated factors (TRAFs). Protein complementation is a novel technique to examine protein-protein interaction through the assembly of functional fluorescent proteins or enzymes from inactive fragments. A previous study in our lab demonstrated the use of bimolecular fluorescence complementation (BiFC) to study the assembly of the LMP1 signaling complexes within the plasma membrane of mammalian cells. In the present study, LMP1 was used as bait in a genome-wide BiFC screen with an enhanced retroviral mutagen to identify new LMP1-binding proteins. Our screen identified a novel LMP1-binding protein, transmembrane protein 134 (Tmem134). Tmem134 is a candidate oncogene that is amplified in breast cancer cell lines. Binding, colocalization, and cofractionation between LMP1 and Tmem134 were confirmed. Finally, Tmem134 affected LMP1-induced NF-B induction. Together, these data suggest that BiFC is a unique and novel platform to identify proteins recruited to the LMP1-signaling complex.

Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins

“…VC1 is an inducible exon trap vector which splices to cellular exons to create CYFP-tagged proteins encoded by genes in which the first base of the exon is the first base of the codon triplet. The various vectors used in the current study were described previously (29,30) and included wild-type LMP1 expression vectors LMP1-NYFP (with a C-terminal NYFP tag), LMP1-CYFP (with a C-terminal CYFP tag), LMP1-NYFP-TH (where TH represents an inducible retrovirus), and M3-LMP1 (where M3 represents an N-terminal triple myc tag). Previously described mutant LMP1 vectors with Cterminal NYFP tags were also used and included LMP1-A5-NYFP (a CTAR1 mutant with 5 A residues [A5]), LMP1-Y384G-NYFP (a CTAR2 mutant), and LMP1-A5-Y384G-NYFP (a CTAR1/CTAR2 mutant).…”

“…CYFP-tagged proteins from the screen were subcloned from cDNA that had been PCR amplified with primers CYFP= and T7= from the BiFCpositive cloned cell lines as described previously (29). Actinin 1, actinin 4, and gelsolin were cloned with the CYFP-hemagglutinin (HA) tags from cDNA into pcDNA3 (Invitrogen).…”

“…Rat-1 Tet-On LMP1-NYFP bait cells were cloned by limiting cell dilution in 96-well plates. Clones were screened for Doxdependent LMP1 expression by In-Cell Western blotting according to the manufacturer's directions (LI-COR) and flow cytometry as previously described (29). Bait cells were infected with the ERM vector VC1 at a multiplicity of infection of 0.3 and selected with Neo, Hygro, and Puro.…”

“…Clone identification was performed as previously described (29). Total RNA was purified from induced VC1 clones and treated with DNase (Qiagen).…”

“…Small-scale retrovirus production was accomplished as previously described (29,30,32) by transfection of HEK-293T cells with retrovirus expression vectors with plasmids expressing vesicular stomatitis virus G glycoprotein and gag-pol using the Transit-LT1 transfection reagent. At 24 h posttransfection, the medium was changed and the cells were moved to 33°C.…”