Summary auto-generated
This study examines how peptide antagonists of HIV-1 Tat protein inhibit viral gene expression. The researchers used Tat9-K-biotin, a peptide containing the TAR-binding domain of Tat, which competes with native Tat for binding to TAR RNA in the HIV-1 long terminal repeat. In Jurkat cells stably expressing a chloramphenicol acetyltransferase (CAT) reporter gene, Tat9-K-biotin effectively blocked Tat-dependent CAT protein production in a dose-dependent manner. Northern blot analysis revealed that the antagonist peptide had minimal effects on CAT mRNA levels, suggesting post-transcriptional inhibition. Polysome association studies demonstrated that while Tat normally increases binding of CAT mRNA to polysomes (translationally active complexes), Tat9-K-biotin dramatically reduced this association, causing CAT mRNA to remain primarily in monosome fractions. The peptide did not affect beta-actin mRNA polysome association, indicating specificity for Tat-dependent transcripts. These findings suggest that Tat9-K-biotin antagonizes Tat function at the translational level by preventing ribosomal loading onto mRNA, rather than through transcriptional inhibition.
Key findings
- Tat9-K-biotin peptide antagonist binds TAR RNA and competes with native Tat protein, blocking Tat-dependent CAT protein expression in cultured cells
- The antagonist peptide causes dramatic reduction in CAT protein levels while leaving CAT mRNA levels largely unchanged, indicating post-transcriptional inhibition
- Tat9-K-biotin prevents polysome association of CAT mRNA, redirecting it to monosome fractions and blocking translational initiation
- Tat protein normally increases polysome association of Tat-dependent transcripts, enhancing their translation
- The inhibitory mechanism is specific to Tat-dependent genes, with minimal effects on non-Tat-dependent transcripts
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Abstract
The Tat (trans-activator of transcription) regulatory protein of human immunodeficiency virus (HIV-1) acts by interacting with the TAR RNA domain of nascent viral transcripts and with cellular proteins to increase viral transcription. In Jurkat-derived HCLE-D36 cells, which are stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene expressed from the TAR-encoding long terminal repeat (LTR) of HIV-1, CAT protein expression is dependent on Tat. The Tat9-K- biotin peptide antagonist of Tat binds specifically to TAR RNA and competes with Tat for binding. In the cellular expression system, Tat9- K-biotin reduces Tat-dependent CAT expression. However, while the Tat antagonist greatly reduces CAT protein production and polysome association of CAT mRNA, it has little effect on CAT mRNA levels, suggesting that the antagonist works at the post-transcriptional level.