MEMBRANE ASSOCIATION OF POLYOMAVIRUS MIDDLE-T ANTIGEN IN AN IN-VITRO SYSTEM

Polyomavirus-infected cells express three proteins in the early phase of the lytic cycle, the so-called tumor antigens. Two of them, large- and middle-T antigens, are also required for virus-mediated transforma tion of primary cells, while middle-T alone is sufficient to transform established cells in culture. Cell transformation by middle-T is stri ctly dependent on the ability of this protein to associate with cellul ar enzymes like members of the Src family of tyrosine kinases, a phosp hatidylinositol 3-kinase, phosphatase 2A and SHC, an adapter protein l inking GDP/GTP exchange factors to tyrosine kinase receptors. A carbox y-terminal stretch of 22 hydrophobic amino acids is required for targe ting middle-T and associated proteins to cellular membranes. Here we s how in an in vitro system that middle-T fusion proteins carrying an am ino-terminal hemagglutinin leader sequence are capable to bind to and enter the lumen of dog pancreas microsomes supporting the concept that the carboxy-terminus of middle-T is an authentic membrane-targeting d omain. Furthermore, wild-type middle-T, but not a truncated protein la cking the putative membrane anchor, specifically associates with artif icial lipid bilayers.