TRANSPOSON-MEDIATED RANDOM INSERTIONS AND SITE-DIRECTED MUTAGENESIS PREVENT THE TRAFFICKING OF A MOUSE MAMMARY-TUMOR VIRUS SUPERANTIGEN

Mouse mammary tumor viruses (MMTVs) encode superantigens (Sags) which are critical to the life cycle of infectious virus and can mediate ext ensive deletion of T lymphocytes when expressed by endogenous provirus es. Little is known about the structure, intracellular trafficking, or nature of Sag association with major histocompatibility (MHC) class I I products. In order to gain a better understanding of Sag structure-f unction relationships, we extensively mutagenized this type II glycopr otein using two different approaches: transposon-mediated random in-fr ame insertion mutagenesis and site-directed mutagenesis targeting clus ters of charged residues. We find that 31 codon insertions are infrequ ently tolerated in Mtv-7 Sag, with just 1 of 14 insertion mutants func tionally presented on the surface of B cells. Surprisingly, similar ef fects were observed with Sag mutants with substitutions at pairs of ch arged residues; only 2 of 6 mutants trafficked to the plasma membrane and stimulated T cells, 1 with a temperature-sensitive phenotype. The data suggest that the nonfunctional Mtv-7 Sag mutants are stringently retained in the endoplasmic reticulum due to conformational defects ra ther than disrupted interactions with MHC class II, thus identifying c harged amino acids critical to the structural stability of viral super antigens. (C) 1998 Academic Press.