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Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral
D Harrich1, G Mavankal, A Mette-Snider
1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA.
Journal of Virology
|August 1, 1995
Summary
Compensatory mutations in the human immunodeficiency virus type 1 (HIV-1) TAR element can restore Tat transactivation, even with altered RNA structures. This suggests nascent RNA, not just the final structure, is key for Tat activation.
Area of Science:
- Molecular Biology
- Virology
- RNA Structure and Function
Background:
- The TAR element in HIV-1's long terminal repeat is crucial for gene expression and replication, mediated by the Tat protein.
- Mutations in TAR RNA structures (stem, bulge, loop) significantly impair HIV-1 gene expression and replication.
- Previous studies showed 200- to 5,000-fold reductions in gene expression with TAR mutations.
Purpose of the Study:
- To investigate the molecular mechanisms behind spontaneous restoration of HIV-1 gene expression and replication in cells with mutated TAR elements.
- To identify compensatory mutations within the TAR element that restore Tat transactivation.
- To elucidate the structural requirements of TAR RNA for effective Tat binding and activation.
Main Methods:
- Characterization of Jurkat cell lines infected with TAR element mutant viruses.
- Analysis of viral DNA sequences from revertant viruses using PCR.
- Gel retardation assays to assess Tat protein binding to in vitro-transcribed revertant TAR RNAs.
- Transfection assays using chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs with original and revertant TAR elements.
Main Results:
- Revertant viruses retained original TAR mutations but acquired compensatory mutations.
- Recombinant Tat protein bound more effectively to revertant TAR RNAs than original TAR mutants.
- Constructs with revertant TAR elements showed strong Tat activation, unlike those with original TAR mutations.
- Altered TAR RNA structures, differing from wild-type, were still capable of robust Tat activation.
Conclusions:
- Compensatory mutations within the TAR element can restore Tat-mediated transactivation despite altered RNA secondary structures.
- The study implicates nascent RNA structures, rather than fully formed TAR RNA, as the primary target for Tat activation.
- These findings refine our understanding of critical sequences and structures in TAR RNA essential for Tat activation.