Targeting RNA-protein interactions within the human immunodeficiency virus type 1 lifecycle

Abstract

RNA-protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA-protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5'-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by ¹H NMR spectroscopy.

Figure 1

Structure of the core Ψ-packaging domain of HIV-1. Schematic representation of the in vitro biophysical assay. Attached to the 5′ and 3′ ends of stem loop 3 (SL3) are the TET fluorophore and blackhole quencher (BHQ1), respectively, which form a destabilization assay in the presence of Gag. Small molecules that inhibit the destabilation of SL3 are taken forward, after validation, to a viral replication assay.

Figure 2

Biological activity of NSC260594. (A) 24 h postinfection the expression of β-galactosidase within the TZM-bl cell were imaged using X-Gal (blue spots in images). (B) Viability of the 293T cells (blue line, 50% inhibition (CC₍₅₀₎ = N/A)), viral production from transfected 293T (green line, 50% inhibition (p24₍₅₀₎ = 11.3 ± 3.4 μM)) and infectivity of harvested viral particles (red line 50% inhibition (IC₍₅₀₎ = 4.5 ± 1.8 μM)) in the presence of different concentrations of NSC260594.

Figure 3

Interaction between the structural analogue of SL3 (WT-3) and the two small molecules, NSC260594 and ellipticine, as monitored by ¹H NMR. (A) Secondary structures of the WT-3 hairpin. In gray are reported the nucleotides added to SL3 in order to have a well-defined secondary structure suitable for NMR spectroscopy and in red the G9, G10, and G12 guanines constituting the tetraloop of SL3. (B) 3-D model of the HIV-1 nucleocapsid–WT-3 complex (PDB 1A1T) depicting the protein and the RNA hairpin with a green and white ribbon, respectively. The G9, G10, and G12 guanines essential for binding to the HIV-1 nucleocapsid are represented in red. (C) WT-3 ¹H NMR titration experiments with an increasing amount of the compounds NSC260594 and ellipticine. The imino protons between 12 and 14.5 ppm (blue region) are attributed to the Watson–Crick H-bonded base pairs of the stem of the hairpin structure and imino protons between 10 and 11 ppm (red region) are attributed to the WT-3 loop G bases. Red stars (★) highlight the alteration of the G9, G10, and G12 imino signals, and black diamonds (⧫) highlight peaks attributed to ellipticine.