Analysis oftrans-Dominant 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

Abstract

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.

Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense

“…18,19,[40][41][42][43][44] Clinical trials using transduced or transfected primary T cells have also shown a protective advantage for cells expressing TdRev. 5,8 In these experi-…”

“…To produce TdRev, we altered the nucleotide sequence of the second Rev exon, such that four amino acids of the Rev C-terminal region are changed (LERLTL to DLRDTD), resulting in a transdominant negative Rev mutant protein. 18 In contrast to HIV-1 where Rev is negatively self-regulated, expression of TdRev in vector pTRG should not inhibit its own synthesis as TdRev does not prevent RNA splicing. As a final component of the pTRG vector, an EGFP gene (or Neo gene in later experiments) was inserted in the place of nef.…”

“…Plasmid pSRD3 encodes a transdominant negative mutant of Rev (TdRev), isolated by PCR from plasmid LR DLDD SN, 18 under the control of the SV40 promoter. 27 A PCR fragment that contains the mutations that converts Rev into TdRev was amplified from pSRD3 using primers 5'-CGATTCTAGAACGGATCCTGAGCA CTTAT-3' and 5'-GATTCTAGAATCGACTAT TCTTTAGCTCCTGAC-3', digested with XbaI and cloned into the XbaI restriction site of vector pLITMUS29-⌬BamHI to obtain plasmid pTRA10.…”

“…[13][14][15][16] Transdominant negative mutants of Rev (TdRev) are one of the best inhibitory anti-HIV genes. [17][18][19] The consti-…”

Improved titers of HIV-based lentiviral vectors using the SRV-1 constitutive transport element

“…6,7 Expressing such anti-HIV genes in HIV-based vectors would require the use of a Rev-independent system to produce significant quantities of vector. Several attempts have been made to develop Rev-independent lentiviral vectors based on HIV-1 or SIV-1.…”