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 extensive deletion of T lymphocytes when expressed by endogenous proviruses. Little is known about the structure, intracellular trafficking, or nature of Sag association with major histocompatibility (MHC) class II products. In order to gain a better understanding of Sag structure-function relationships, we extensively mutagenized this type II glycoprotein using two different approaches: transposon-mediated random in-frame insertion mutagenesis and site-directed mutagenesis targeting clusters of charged residues. We find that 31 codon insertions are infrequently tolerated in Mtv-7 Sag, with just 1 of 14 insertion mutants functionally presented on the surface of B cells. Surprisingly, similar effects were observed with Sag mutants with substitutions at pairs of charged 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 rather than disrupted interactions with MHC class II, thus identifying charged amino acids critical to the structural stability of viral superantigens.
McMahon
C W
CW
Department of Immunology, University of Washington, Seattle 98195, USA.
Traxler
B
B
Grigg
M E
ME
Pullen
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004251:DNA Transposable Elements; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D007424:Intracellular Fluid; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D018089:Superantigens / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013696:Temperature; D014407:Tumor Cells, Cultured
D000956:Antigens, Viral; D004251:DNA Transposable Elements; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; C095378:Mtv-7 superantigen, Mouse mammary tumor virus; D018089:Superantigens