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. 1997 Nov 11;94(23):12592-7.
doi: 10.1073/pnas.94.23.12592.

The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB

K M Izumi  1 , E D Kieff
Affiliations

The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB

K M Izumi et al. Proc Natl Acad Sci U S A. .

Abstract

The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth.

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Figures

Figure 1
Figure 1
Diagram of LMP1. The Flag epitope was introduced at the amino terminus (NH2). LMP1 residues 187, 231, 352, and 386 are noted. LMP1 constitutively aggregates in the plasma membrane. TES1 engages TRAFs to drive initial B lymphocyte proliferation and induce low-level NF-κB activation. TES2 engages TRADD to enable efficient long-term lymphoblastoid cell outgrowth and mediate high-level NF-κB activation through TRAF2.
Figure 2
Figure 2
Characterization of LMP1 in infected LCLs. (A) Southern blot analysis of EBV recombinant infected LCLs. DNA cut with SacI and MluI was Southern blot-probed with EBV MluI DNA (nucleotide 167,129 to 169,560), which comprises LMP1 DNA. A 2.4-kb DNA from P3HR-1 cells and a wild-type EBV-transformed LCL is hybridized by the probe. In LCLs F-LMP1 1.1, 1.2, and 2.1, which are infected with an F-LMP1 recombinant from F-LMP1–1 or F-LMP1–2 LCL, a SacI site near the Flag codons results in a 2.3-kb DNA whereas the 0.16-kb DNA ran off. In coinfected LCLs F-LMP1-ID 1–4 or in singly infected LCLs F-LMP1-FFD 1.1–1.6, which are infected with an F-LMP1-FFD recombinant from F-LMP1-FFD-1 LCL, F-LMP1-ID, and F-LMP1-FFD DNAs, have a second SacI site near the last codon, resulting in 1.2- and 1.1-kb DNAs whereas the 0.16-kb DNA ran off. The 2.4-kb DNA in LCLs F-LMP1-ID 1–4 is P3HR-1 LMP1 DNA. Markers in kb are at left. (B) Immunoblot analysis of LMP1. Proteins from 5 × 104 cells were size-separated, blotted to filters, and probed with antibody to Flag. A 60-kDa band in LCLs F-LMP1 1.1, 1.2 and 2.1, F-LMP1-ID 1–4, and F-LMP1-FFD 1.1 and 1.2 is Flag-LMP1 (wild type or mutant). A standard in kDa is noted at left. (C) Immunofluorescent staining of cells with antibody to Flag. (C1) F-LMP1-ID-1, an LCL coinfected with P3HR-1 and F-LMP1-ID recombinant. (C2) F-LMP1 1.1, an LCL infected with F-LMP1 recombinant only. (C3) P3HR-1 that expresses LMP1. (C4) LCL infected with wild-type EBV that expresses LMP1 without Flag.
Figure 3
Figure 3
(A) Specific coprecipitation of TRADD with F-LMP1. 293 cells were electroporated with pRK5-myc-TRADD and pSG5-based F-LMP1 or F-LMP1-ID vector DNAs. Nonidet P-40-solubilized proteins were incubated with M2 antibody to Flag coupled to beads (Kodak). M2 beads were washed, and precipitated proteins were solubilized and immunoblot-analyzed with 9E10 antibody to myc-TRADD or S12 antibody to LMP1. Unfractionated cell proteins are inputs, and proteins not precipitated with M2 beads are unbound. Percentages indicate fraction of total sample. (B) Coprecipitation of TRADD with F-LMP1. LCLs (1.5 × 108 cells) infected with an F-LMP1 EBV recombinant or with a wild-type LMP1 EBV recombinant were solubilized in buffer containing Brij 58 and incubated with M2 beads. Precipitated proteins were solubilized and Western blot-analyzed with antisera to TRADD (Santa Cruz Biotechnology) or S12 antibody to LMP1. Unfractionated cell proteins are inputs and proteins not precipitated with M2 beads are unbound. Percentages indicate fraction of total sample.
Figure 4
Figure 4
(A) F-LMP1 expression in 293 cells activates NF-κB. Five million cells were electroporated with 5 μg of pSG5 or pSG5-LMP1 vector DNA, 2.5 μg of 3×-κB-L luciferase reporter DNA, and 2.5 μg of glucokinase promoter/β-galactosidase reporter DNA as control. Residues of LMP1 are indicated within parenthesis, Δ indicates deletion of residues 187–351, FFD or ID indicates replacement of Y384YD386, and P > A indicates P379 replaced with A within a potential TRAF binding site. Cell lysates were analyzed for luciferase (Promega) and β-galactosidase (Tropix, Bedford, MA) on an Optocomp I luminometer. Immunoblots probed with M5 antibody to Flag (data not shown) indicated that LMP1 expressions levels were similar in the transfected cells. (B) TRADD and F-LMP1 synergize to activate NF-κB. 293 cells were electroporated with the indicated amounts of pSG5 or pSG5-F-LMP1 or F-LMP-ID vector DNA and with pRK5 or pRK5-mycTRADD vector DNA, 2.5 μg of 3×-κB-L luciferase reporter DNA, and 2.5 μg of glucokinase promoter/β-galactosidase reporter DNA as control. Lysates were analyzed for luciferase (Promega) and β-galactosidase (Tropix) on an Optocomp I luminometer. Immunoblots probed with M5 antibody to Flag (data not shown) indicated that LMP1 expressions levels were similar for the indicated amounts of DNA in the respective transfected 293 cells.
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