ANALYSIS OF TRANSDOMINANT MUTANTS OF THE HIV TYPE-1 REV PROTEIN FOR THEIR ABILITY TO INHIBIT REV FUNCTION, HIV TYPE-1 REPLICATION, AND THEIR USE AS ANTI-HIV GENE THERAPEUTICS

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 Re v mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral ex pression vectors and analyzed for their ability to inhibit Rev-mediate d gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-d ependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expre ssion was tested in the HeLa-T4 cell line and was also shown to be inh ibited by the trans-dominant Rev mutants. Retroviral vector producer c ell lines were constructed and used to transduce Rev trans-dominant ge nes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitore d by Northern blot analysis and in situ hybridization. SupT1 cells exp ressing either a Rev point mutant or the frameshift mutant showed grea tly reduced HIV-1 mRNA accumulation and the Rev-dependent singly splic ed and unspliced HIV-1 mRNAs were reduced. The kinetics of viral repli cation following challenge of Rev trans-dominant-engineered SupT1 cell s with both HIV-1 IIIB and MN strains was significantly reduced and ce lls were protected from viral lysis. Viruses that emerge late in infec tion 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.