All three domains of the Epstein-Barr virus-encoded latent membrane protein LMP-1 are required for transformation of rat-1 fibroblasts.
LMP-1, the Epstein-Barr virus latent membrane protein 1, is the only protein encoded by the virus that has been shown to have the properties of a transforming oncogene in rodent fibroblasts such as Rat-1 cells. LMP-1 is phosphorylated and proteolytically cleaved in Rat-1 cells in a manner similar to that seen in human lymphocytes. In this study, we demonstrate that all three major domains of LMP-1 (N-terminal, transmembrane, and C-terminal domains) are required for the ability to transform Rat-1 cells in culture, as assayed by loss of contact inhibition. This study is the first demonstration of a functional role for the C-terminal domain of LMP-1. Our analysis suggests that there are at least three distinct regions of the C terminus involved in signalling. Amino acids 306 to 334, which generate a toxic signal in the absence of amino acids 334 to 364, and the last 23 amino acids, 364 to 386, are essential for transformation. Biochemical analysis of the LMP-1 mutants with the three domains deleted indicate that the mutant N-terminal with the domain deleted is phosphorylated normally but is inefficiently cleaved compared with the wild-type LMP-1. The mutant with the transmembrane domain deleted is also phosphorylated but is not cleaved, showing that phosphorylation of LMP-1 does not require membrane association. The nontransforming mutant with the C-terminal domain deleted that lacks the last 23 amino acids is phosphorylated and cleaved. Therefore, these processing events alone are insufficient to generate a transforming signal.
Moorthy
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RK
Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Thorley-Lawson
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DA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004252:DNA Mutational Analysis; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection; D014170:Transformation, Genetic; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D009856:Oncogene Proteins, Viral; D011498:Protein Precursors; D021382:Protein Sorting Signals; D014763:Viral Matrix Proteins