Construction of a selectable nef-defective live-attenuated human immunodeficiency virus expressing Escherichia coli gpt gene

We have developed a replication-competent human immunodeficiency virus (HIV ) carrying a selective marker that can be used in vivo. This recombinant vi rus (Z6 Delta nef gpt) was generated by replacing the 5' half of the HIV ne f gene with the Escherichia coil guanine phosphoribosyl transferase gene (g pt). This new vector can express the gpt product on infection and works as a positive selective marker for mycophenolic acid (MPA) resistance, a poten t immunosuppressive drug used in organ rejection therapy. Conversely, gpt e xpression also served as a negative selectable marker, since its intracellu lar expression induces host-cell susceptibility to 6-thioxantine (6-TX), a nucleotide analog that is toxic to the infected cell under these conditions . In this manner, we could suppress the recombinant virus replication throu gh 6-TX selection in both transformed cells and primary human peripheral bl ood mononuclear cells (PBMCs), suggesting the vector's potential as a model for a new live-attenuated vaccine approach against HIV. (C) 2000 Academic Press.