Mutational analysis of the ligand-binding domain of M-T2 protein, the tumor necrosis factor receptor homologue of myxoma virus.
The myxoma virus-encoded M-T2 protein shares extensive sequence homology with the ligand-binding domains of the TNF receptors (TNFRs) and has been shown to bind and inhibit rabbit TNF-alpha with affinities similar to those of TNF-alpha with cellular receptors. Here we show that M-T2 protein is secreted from infected cells as an N-linked glycoprotein, with both complex and hybrid or high mannose oligosaccharide chains. Since amino acid homology between M-T2 and cellular TNF receptors is limited to the four N-terminal cysteine-rich domains (CRDs), various M-T2 C-terminal truncations were created in recombinant vaccinia virus vectors. C-terminal deletions that include truncations up to the middle of the fourth CRD effectively bound and inhibited rabbit TNF-alpha. In contrast, removal of any one of the first three CRDs resulted in a mutant M-T2 protein incapable of binding or inhibiting rabbit TNF-alpha. The C-terminal portion of M-T2, which is not homologous to the cellular TNFRs, appears to be important for efficient secretion of M-T2 from infected cells, since all the C-terminal truncations, including a truncation removing only the last 24 amino acids, were effectively retained as intracellular proteins that were still capable of binding and inhibiting rabbit TNF-alpha. We conclude that the first three CRDs of M-T2 fulfill the same ligand-binding function as the cellular TNFRs, and the nonhomologous C-terminal region participates in protein trafficking of M-T2 in virus-infected cells.
Department of Biochemistry, University of Alberta, Edmonton, Canada.
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M
Schreiber
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McFadden
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D018604:Epitope Mapping / Q000379:methods; D005822:Genetic Vectors / Q000378:metabolism; D006031:Glycosylation; D008024:Ligands; D009233:Myxoma virus / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors; D014616:Vaccinia virus / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
D008024:Ligands; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins