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.