Inhibition of Both HIV-1 Reverse Transcription and Gene Expression by a Cyclic Peptide that Binds the Tat-Transactivating Response Element (TAR) RNA

Abstract

The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC ₅₀ ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (−) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.

Author Summary

The HIV-1 transactivator protein (Tat), together with the elongation factor P-TEFb binds to an HIV-1 RNA secondary structure in the 5′-UTRs of nascent viral mRNAs (TAR) and promotes transcription elongation. This process has been an attractive target for drug development but previous inhibitors that bind either Tat or TAR have been plagued by poor inhibition of virus replication, limited cell penetration, and off-target effects. In this article, we describe a series of rationally designed cyclic peptides that block Tat-TAR interactions. L50, the most potent of these compounds, inhibits a wide range of HIV-1 strains from around the world. Remarkably, L50 inhibits two distinct steps in the HIV-1 lifecycle. As expected, L50 inhibits Tat-dependent HIV-1 transcription, but the majority of its anti-HIV activity is due to a block in reverse transcription, i.e. synthesis of the proviral DNA from the RNA genome. L50 inhibition of reverse transcription reveals an important role for TAR RNA during reverse transcription as well as providing one of first examples of a drug with a dual mechanism of action.