We are exploring an approach to gene therapy for HIV infection based on blocking the function of viral and cellular proteins responsible for activation of viral gene expression. The focus is on blocking those functions of the viral protein tat which are essential for trans- activation, and on the trans-activator responsive (TAR) element RNA which is critical to this process. We will engineer lymphoid cell lines that express regulatable levels of TAR containing RNA in order to examine whether excess TAR can block trans-activation and virus production in infected cells by competing with newly synthesized HIV LTR RNA for the trans-activation machinery. If so, human peripheral blood cells will be constructed which overexpress TAR and then used to examine the effects of TAR overproduction on HIV infection using the mouse/human chimera model for AIDS being developed by Dr. J Belmont (Core C). We also work to develop rational approaches to the design of alternative TAR analogs which may be more effective in the gene therapy strategies outlined above and will allow us to further explore the chemical basis of the trans-activation process. One approach involves a determination of the precise structural features in TAR RNA necessary for its interaction with the trans-activation machinery. We will use novel spectroscopic approaches, employing new fluorescent derivatives of TAR RNA, to explore the structure and dynamics of TAR RNA and to monitor directly its interaction with tat. A combination of chemical, enzymatic, and genetic techniques will be used to construct base- specific TAR mutants; analogs in which single substituents (located on individual nucleotides contained in the trans-activation responsive stem- loop structure) have been removed or modified, thereby eliminating the ability to form specific hydrogen bonds. Spectroscopic studies will allow us to dissect the individual contributions of each of the deleted substituents to the energetics and dynamics of TAR function. Coupled to this approach will be the development of affinity labeling TAR derivatives useful in the identification of residues in tat which interact directly with specific nucleotides in the RNA. The information gained from these studies will be used to design TAR analogs that bind more tightly to the trans-activation machinery, and that may therefore be more effective in blocking trans-activation and virus production.

Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
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Ansari-Lari, M A; Donehower, L A; Gibbs, R A (1995) Analysis of human immunodeficiency virus type 1 integrase mutants. Virology 211:332-5
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Simoneaux, D K; Fletcher, F A; Jurecic, R et al. (1995) The receptor tyrosine kinase-related gene (ryk) demonstrates lineage and stage-specific expression in hematopoietic cells. J Immunol 154:1157-66
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Echetebu, C O; Rhim, H; Herrmann, C H et al. (1994) Construction and characterization of a potent HIV-2 Tat transdominant mutant protein. J Acquir Immune Defic Syndr 7:655-64
Ansari-Lari, M A; Gibbs, R A (1994) Analysis of HIV type 1 reverse transcriptase expression in a human cell line. AIDS Res Hum Retroviruses 10:1117-24
Rhim, H; Rice, A P (1994) Exon2 of HIV-2 Tat contributes to transactivation of the HIV-2 LTR by increasing binding affinity to HIV-2 TAR RNA. Nucleic Acids Res 22:4405-13
Rhim, H; Echetebu, C O; Herrmann, C H et al. (1994) Wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli as fusions with glutathione S-transferase. J Acquir Immune Defic Syndr 7:1116-21
Lewis, D E; Gibbs, R A (1993) Detection and significance of HIV sequences in HIV infection. Prog Med Virol 40:19-47

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