The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.