Functional testing of a bicistronic retroviral vector for intracellular peptide production.

Cytotoxic T cells generally recognize short 8-mer to 11-mer peptides in association with major histocompatibility complex (MHC) class I molecules on the surface of the antigen presenting cells (APCs). Usually, these peptides are produced in the cytosol of the APCs by proteolytic degradation of cytosolic proteins. The peptides are subsequently transported first to the endoplasmatic reticulum (ER), then to the cell surface and are bound to the MHC class I molecule. In the study of these events, transient expression from recombinant vaccinia viruses encoding short immunogenic peptides has been shown to lead to peptide presentation on the MHC class I molecules (2,6). In this work, a novel retroviral vector for expression of peptides has been developed, which potentially could be used for intracellular expression of large random libraries of short peptides. Such retroviral vector-based cellular libraries can be prepared in the way that each cell only expresses a single peptide species derived from the library–the “one cell-one peptide” principle. Due to the replication cycle of the virus, expression of such a library using vaccinia viruses would have some limitations, because it will result in many different peptides being produced transiently in single cells. Vaccinia viruses infect most mammalian cells and replicate transiently in the cytoplasm where they inhibit host-protein synthesis and elicit a rapid cytopathic effect on the host cell (16). A bicistronic retroviral vector encoding a single RNA transcript was constructed using standard recombinant techniques (Figure 1). The vector backbone is derived from the murine retrovirus Akv (13). Downstream from the packaging signal, the vector contains a polylinker enabling conventional cloning of peptide-encoding sequences or insertion by non-polymerase chain reaction (PCR)-mediated overlap extension (9). Following the polylinker, an internal ribosome entry site (IRES) derived from the encephalomyocarditis (EMC) virus is inserted (10). This IRES directs translation of a neo gene conferring G418 resistance to transduced cells. To ensure that the IRES cassette was functional in a retroviral vector context, the sequences encoding the peptide were exchanged with the phleomycin-resistence gene (pUT649; CAYLO, Toulouse, France). After transduction with more than 105 NIH-3T3 cells of a diluted stock of this vector, 38 colonies resistant to phleomycin and 21 colonies resistant to G418 were isolated. All phleomycin-resistant colonies were G418 resistant, and all G418-resistant colonies were phleomycin resistant, indicating that both resistance genes were translated from the same vector’s RNA transcript. As a test system for evaluating whether the vector could express such small peptides, we used a functional MHC-class I presentation assay. Two vectors encoding the peptides SIINFEKL and TPHPARIGL, respectively, were constructed. These peptides correspond to two previously described MHC-class-I-restricted T-cell epitopes derived from ovalbumin and Escherichia coli β-galactosidase, respectively (1,5). The peptides bind specifically to the mouse Kb and Ld MHCclass-I molecules, respectively. The vector harbors a methionine start codon in front of the peptide-coding regions and a stop codon immediately after. APCs of relevant MHC-I haplotypes were transduced using the Psi-2 packaging cells (14) with standard retroviral vector transduction techniques (Figure 1) (12). The APCs were selected for G418 resistance, and resistant colonies were pooled and assayed for peptide presentation on the relevant MHCclass-I molecules. Mixing the APCs with specific T-cell hybridomas causes Benchmarks