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

Abstract

We have developed a replication-competent human immunodeficiency virus (HIV) carrying a selective marker that can be used in vivo. This recombinant virus (Z6 Delta nef gpt) was generated by replacing the 5' half of the HIV nef gene with the Escherichia coli guanine phosphoribosyl transferase gene (gpt). This new vector can express the gpt product on infection and works as a positive selective marker for mycophenolic acid (MPA) resistance, a potent immunosuppressive drug used in organ rejection therapy. Conversely, gpt expression also served as a negative selectable marker, since its intracellular 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 through 6-TX selection in both transformed cells and primary human peripheral blood mononuclear cells (PBMCs), suggesting the vector's potential as a model for a new live-attenuated vaccine approach against HIV.