The chronicity of infection by the human immunodeficiency virus (HIV) calls for therapeutic regimens that offer sustained antiviral effects, such as gene therapy. Recent studies have demonstrated that expression of HIV mutant transdominant proteins, RNA decoys, and ribozymes efficiently inhibited HIV replication. We have previously shown that an RNA decoy (stem-loop II of the Rev response element of HIV type 1 [HIV-1], named SL2) and a ribozyme (Rz) targeting the U5 region of the HIV-1 5' long terminal repeat (LTR), combined in a fusion molecule, was more efficient in inhibiting HIV-1 replication than the ribozyme or the decoy alone. In this study, we expressed this fusion molecule in a retrovirus-based double-copy vector to obtain higher expression of this molecule. Furthermore, we inserted a sequence internally to drive expression of another fusion molecule with a ribozyme targeting the env/rev region linked to SL2 to obtain a triple-copy vector. These multigene antiviral vectors were subsequently transduced or transfected into human CD4+ T cells (Molt-4). Results showed that the translocation of the SL2-Rz cassette from the 3' to the 5' LTR occurred in 80% of the transduced cells. The numbers of ribozyme RNA transcripts, estimated by competitive-quantitative reverse transcription (RT)-PCR, were 1.2 x 10(5), 1.2 x 10(4), and 1.5 x 10(3) copies per cell for the triple-copy, double-copy, and single-copy vectors, respectively. Cell challenge with multiple subtypes of HIV-1 (clades A to E) showed commensurate levels of virus inhibition for the three vectors. This study suggests that the combination of multiple anti-HIV genes, such as ribozymes and decoys, targeting multiple sites of HIV RNA and expressed at high levels are promising for the treatment of HIV-1 infection.