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. 1997 Feb 18;94(4):1447-52.
doi: 10.1073/pnas.94.4.1447.

The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation

K M Izumi  1 , K M Kaye, E D Kieff
Affiliations

The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation

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

Abstract

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs). We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185-211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1 delta185-211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1 delta185-211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.

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Figures

Figure 1
Figure 1
Structure of LMP1. The Flag epitope was introduced at the LMP1 amino terminus (NH2). LMP1 residues 185, 211, 231, and 386 are noted. The single TRAF binding site and the two NF-κB-inducing domains are indicated.
Figure 2
Figure 2
PCR analysis of LMP1 DNA. (A) LMP1 DNAs in supernatants from lytically infected LCLs coinfected with P3HR-1 and Flag LMP1 recombinant EBV (F-L2 in lane 1) or P3HR-1 and Flag LMP1Δ185–211 recombinant EBV (F-ΔL1 to F-ΔL5 in lanes 2–6) were analyzed by PCR with ΔL5′ (5′-ctctattggttgatctcctttgg-3′) and 3′L315 (5′-attgtggagggcctccatcatttc-3′). Control DNAs pL Flag LMP1 (lane 7) and pL Flag LMP1Δ185–211 (lane 8), and DNA from EBV-negative BJAB cells (lane 9) were also analyzed. Size standards in base pairs are noted to the left. (B). PCR detection of wt LMP1 DNA with 5′L1 (5′-cacgcgttactctgacgtagccg-3′) and WTND (5′-tcctcgagggggccgtcgc-3′). Lanes 1–7 contain 104 IB4 cells (4 genomes per cell) serially 10-fold diluted with 104 EBV-negative BJAB cells. The endpoint lies after lane 5 for a sensitivity of 4 wt LMP1 DNAs per 104 cells. wt LMP1 DNA is in LCL F-L2 (lane 9) but not in LCL F-L1 (lane 8) or LCLs F-L2.1 and F-L2.2 (lanes 10 and 11). Size standards in base pairs are to the left.
Figure 3
Figure 3
Southern and Western blot analyses. (A) DNA from LCLs was cut with SacI and MluI, size-separated, and probed by Southern blotting with an EBV MluI DNA (nucleotides 167,129–169,560) which comprises LMP1 DNA. A 2.4-kb band from P3HR-1 (lane 1) and a wt EBV transformed LCL (lane 3) is recognized by this probe. In LCL F-L1 (lane 2), a SacI site near the Flag codons results in a 2.3-kb DNA, whereas the 0.16-kb band ran off the gel. In coinfected LCLs F-ΔL1 to F-ΔL5 (lanes 5–9), Flag LMP1Δ185–211 DNAs have a second SacI site near the last LMP1 codon, resulting in 1.2- and 1.1-kb DNAs, while the 0.16-kb DNA ran off the gel. The 2.4-kb DNA in lanes 5–9 is wt LMP1 DNA. DNA standards are noted on the left. (B) Denatured proteins from 5 × 104 LCL cells were size-separated, blotted to filters, and probed with M5 Flag antibody (Kodak). No signal is detected in P3HR-1 cells (lane 1), in a wt EBV-transformed LCL (lane 3), or in EBV-negative BJAB cells (lane 4). Flag LMP1 (60 kDa) is detected in LCL F-L1, whereas Flag LMP1Δ185–211 (57 kDa) is detected in LCLs F-ΔL1 to F-ΔL5 (lanes 5–9). A protein standard is noted on the left. (C) The same blot was stripped of antibody and reprobed with S12 monoclonal antibody to LMP1. All cells except EBV-negative BJAB (lane 4) demonstrate 60-kDa LMP1 or Flag LMP1.
Figure 4
Figure 4
Immunofluorescent staining of cells with M5 antibody to the Flag epitope. (A) F-ΔL1, an LCL coinfected with P3HR-1 and Flag LMP1Δ185–211 EBV recombinant. (B) F-L1, an LCL infected with Flag LMP1 EBV recombinant only. (C) P3HR-1. (D) LCL infected with wt EBV.
Figure 5
Figure 5
Flag LMP1Δ185–211 does not engage TRAF3 or TRAF1 in an LCL. F-L1 cells (107), an LCL infected with Flag LMP1 EBV recombinant, or 4 × 107 F-ΔL1 cells, an LCL coinfected with Flag LMP1Δ185–211 EBV recombinant and P3HR-1, were disrupted in Nonidet P-40 buffer. Part of the input cell lysate was withheld, and the rest was mixed with M2 antibody coupled to beads. Unbound proteins were separated from beads by centrifugation. Proteins coprecipitating with Flag LMP1 or Flag LMP1Δ185–211 were analyzed by Western blotting with anti-TRAF3 sera (Santa Cruz Biotechnology, top row), with S12 antibody to LMP1 (middle row), and with anti-TRAF1 sera (Santa Cruz Biotechnology, bottom row). Percentages indicate the fraction of the total cell lysate loaded into each lane. The positions of TRAF3 (T3), Flag LMP1 (FL), wt LMP1 (L), Flag LMP1Δ185–211 (FΔ), and TRAF1 (T1) are indicated to the left. Mouse Ig heavy chains (middle row, lanes 5 and 6) are just below Flag LMP1 and LMP1.
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