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. 1993 Jun;67(6):3199–3207. doi: 10.1128/jvi.67.6.3199-3207.1993

Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes.

I Bahner 1, C Zhou 1, X J Yu 1, Q L Hao 1, J C Guatelli 1, D B Kohn 1
PMCID: PMC237659  PMID: 8388497

Abstract

trans-Dominant inhibitory mutant versions of the human immunodeficiency virus type 1 (HIV-1) regulatory genes tat and rev have previously been described. We have constructed a series of retroviral vectors to transduce these genes and compare their inhibitory activities. The inhibitory activities were measured with transient transfection assays by using a reporter which expresses an HIV-1 gag-Escherichia coli lacZ fusion protein with strict dependence on coexpression of both tat and rev. Additionally, the vectors were packaged as amphotropic virions and used to stably transduce human CEM T lymphocytes. The transduced CEM cells were challenged with HIV-1, and the effects of the mutant HIV-1 genes were determined by measuring the levels of HIV-1 p24gag produced. A tat gene substituted at amino acid 41 (tatk41a) retained partial trans-activating activity and lacked inhibitory activity. A tat gene with a premature stop codon at amino acid 54 (tat54ter) showed moderate trans-dominant inhibition of the reporter plasmid but failed to significantly inhibit HIV-1 replication. The M10 rev mutant, with a 2-amino-acid substitution, showed strong trans-dominant inhibitory activity both in the reporter plasmid and in the HIV-1 infection assay. The greatest inhibition of HIV-1 growth was seen when M10 was expressed under the transcriptional control of a human cytomegalovirus promoter; slightly less inhibition was achieved when expression of M10 was controlled by the Moloney murine leukemia virus long terminal repeat, and minimal inhibition was seen when the HIV-1 long terminal repeat controlled the M10 gene. These results demonstrate the potential utility of retroviral vectors expressing trans-dominant inhibitory mutant HIV-1 genes for gene therapy approaches to AIDS.

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Selected References

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