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 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.
Izumi
K M
KM
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Kaye
K M
KM
Kieff
E D
ED
D001402:B-Lymphocytes / Q000821:virology*; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D000939:Epitopes; D006412:Hematopoietic Stem Cells / Q000821:virology; D004854:Herpesvirus 4, Human / Q000235:genetics*; D008223:Lymphoma / Q000209:etiology; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D017384:Sequence Deletion; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D048008:TNF Receptor-Associated Factor 3; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D000939:Epitopes; D011506:Proteins; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D048008:TNF Receptor-Associated Factor 3; D014763:Viral Matrix Proteins