Bis-3-Chloropiperidines Targeting TAR RNA as A Novel Strategy to Impair the HIV-1 Nucleocapsid Protein

Abstract

Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem-bulge-loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs' recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro.

Keywords: TAR-RNA; alkylating agents; bis-3-chloropiperidines.

Figure 1

(A) Sequence and secondary structure of oligonucleotide-construct replicating trans-activation response (TAR) RNA which was employed in our assays. (B) Chemical structure of bis-3-chloropiperidines analyzed in this work.

Figure 2

Variations of TAR RNA melting temperature (ΔT_m) in the presence of an increasing concentration of B-CePs 1–5. Reported values are the mean ± standard error of the mean (SEM) of triplicate experiments performed on samples containing 1 μM of oligoribonucleotide and 10, 50 and 100 μM of compound. TAR RNA was folded in a BPE buffer and an incubation of TAR RNA with each compound at 37 °C for 2 h was performed before the analyses. Reference the T_m value for TAR RNA in the absence of compound is 52.9 °C.

Figure 3

Non-denaturing polyacrylamide gel electrophoresis (PAGE) (PAA 12%) in TBE 1× showing the concentration-dependence of the interaction of B-CeP 1 with TAR. Folded TAR hairpin 1 μM was incubated with increasing concentrations of compound (0, 1, 5, 10, 25, 35, 50 μM) for 2 h at 37 °C with a BPE buffer (pH 7.4).

Figure 4

Representative electrospray-ionization mass spectrometry (ESI-MS) spectra of reaction mixtures obtained by incubating TAR (1 μM) with increasing B-CeP 1 (A:0, B:1, C:10, D:50 μM) at 37 °C for 2 h in a BPE buffer (pH 7.4). The spectra B–D show the concentration-dependent reactions induced by B-CeP 1 on TAR. Greater stoichiometries corresponding to combinations of mono- and bi-functional adducts, were detected and indicated in orange in the figure. The spectra were recorded in 150 mM ammonium acetate. Lower intensity signals near free/adducted species consist of typical sodium and ammonium adducts. Only region containing the 5-charge state is shown for the sake of clarity.

Figure 5

(A) Representative region of ESI-MS spectrum obtained after RNAse A digestion of TAR RNA (1 μM) reacted with 10 μM of B-CeP 1. Only the most relevant bridged alkylation products, which are cross-referenced in Table 2 and highlighted in the cartoon on panel B, are indicated in the spectrum. Oligonucleotides products are indicated by the first and last base separated by colon. Product labels indicate the bi-functional alkylation products bridging base-paired regions within the TAR RNA hairpin. (B). Cartoon of the B-CeP 1-induced bi-functional alkylation products bridging base-paired regions within the TAR RNA secondary structure.