Mapping of the ICOS binding surface of murine B7h using an unbiased, cellular library of B7h mutants created by cyclical packaging rescue

Functional studies of immunologically relevant molecules often involve time-consuming generation and cloning of gene mutations prior to introduction into mammalian cells. We describe here an alternative mutagenesis approach that relies solely on transfer of helper-free retroviral supernatants to rapidly create in virtually any cell line of interest a large cellular library that retrovirally expresses a defined number of independent point mutations in a gene of interest. Using this rapid non-cloning approach, we generated a 3T3 cellular library retrovirally expressing 2 × 105 mutants of the murine costimulatory B7h gene. Screening of this unbiased cellular library identified six residues of murine B7h that are critical for binding to the ICOS receptor. These residues are located on the same strands of human B7h that were identified by targeted mutagenesis [Chattopadhyay, K., Bhatia, S., Fiser, A., Almo, S.C., Nathenson, S.G. (2006). Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor-binding site of the protein. J. Immunol. 177, 3920], indicating that the ICOS receptor-binding interface is similar in mouse and human B7h. Based on this proof-of-principle study, CPR-based mutagenesis is applicable to studies of gene function in a variety of mammalian cells.