THE EPSTEIN-BARR-VIRUS LMP1 AMINO-ACID-SEQUENCE THAT ENGAGES TUMOR-NECROSIS-FACTOR RECEPTOR-ASSOCIATED FACTORS IS CRITICAL FOR PRIMARY B-LYMPHOCYTE GROWTH TRANSFORMATION

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 cod ons 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 impor tance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185-211. Thi s mutation eliminates TRAF association in yeast and in lymphoblasts bu t does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells w ith overlapping EBV cosmids, Infection of primary B lymphocytes result ed in cell lines that were coinfected with an LMP1 Delta 185-211 EBV r ecombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is tran sformation defective due to another deletion. Despite the equimolar mi xture of wild-type and mutated LMP1 genes in virus preparations from f ive coinfected cell lines, only the wild-type LMP1 gene was found in 4 12 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1 Delta 185-211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregat ed from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transforma tion.