An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

Abstract

Background: NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays.

Results: Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect.

Conclusions: NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.

Keywords: Antiretroviral drugs; HIV-1; Packaging; RNA structure.

Fig. 1

Viral integration levels determined in the presence and absence of NSC and integration inhibitor. Jurkat cells were pre-treated with NSC [100 µM] or raltegravir [1 µM], and infected with LAI 6 h post-treatment in the presence or absence of 50 µM NSC or 500 nM raltegravir. 24 h post-infection, cells were harvested and subjected to DNA extraction and HIV-1 integration was assayed by Alu-gag PCR followed by quantification of integration events by HIV-1 gag qPCR. The number of integration events relative to LAI-infected cells (WT) in the presence of NSC or raltegravir is shown. For each sample, variations in the amount and quality of cellular DNA input was accounted for by dividing the number of HIV-1 integration events detected by the HIV-1 gag qPCR by the relative actin level detected in the input DNA for the Alu-gag PCR. All samples were then normalized relative to one another such that the average WT value was 1. The average of three samples is shown for each condition. Error bars represent SD. *Statistically significant from WT by Student’s t test, p < 0.05)

Fig. 2

Viral Gag production in the presence and absence of NSC. 293T cells were transfected with HIVΔEnv (WT and NSC) or HIVΔp1ΔEnv (Δp1) and VSV-G expression plasmids. 6 h post-transfection, cells were treated with 50 μM NSC (NSC) or the equivalent quantity of DMSO (WT and Δp1). 24 h post treatment, supernatants and cells were harvested. Equal volumes of samples were electrophoresed on SDS-PAGE gels and immunoblots performed using anti-p24/p55 antibody. Cellular blots were stripped and reprobed with anti-GAPDH antibody. a Results of cellular, supernatant and supernatant:cellular Gag expression levels, quantified using densitometry. Results show an average of four independent experiments, each containing 3–6 replicates (with each replicate examining an independently transfected well of a six-well plate) of each experimental condition. b Anti-Gag Western blot showing intracellular Gag expression levels, and anti-GAPDH control. Two experimental replicates, from independently transfected wells are shown (c) anti-Gag Western blot showing extracellular Gag expression. Two experimental replicates from independently transfected wells are shown

Fig. 3

Viral gRNA levels analyzed by qRT-PCR, in the presence and absence of NSC. 293T cells were treated as in Fig. 2. 24 h post treatment, supernatants and cells were harvested, virions purified from 500 μL supernatant and RNA extracted. a gRNA levels in cells, analyzed by qRT-PCR. Each sample was first normalized against β-actin levels and then normalized against the average wild-type gRNA levels. Data shown are the average of 14 –18 samples taken from four independent experiments. Error bars represent SD. b Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to gRNA levels inside cells. Intracellular levels were first normalized against β-actin; these values were then used to divide the virion RNA levels for each sample. Data were then normalized against the wild-type average. Data are shown as three independent experiments, each containing 4–9 replicates. c Ratio of gRNA levels in purified virions from equivalent volumes of supernatant to cytoplasmic gRNA. Data are representative of two independent experiments. d Ability of virions produced in the presence or absence of NSC to transduce cells, measured by luciferase assay. Error bars represent the SD. *p < 0.05; **p < 0.01 by Student’s t test. Data are representative of two independent experiments

Fig. 4

Effects of NSC on dimerization of viral RNA. In vitro dimerization assays in the presence and absence of 10× molar ratio of NSC. RNA was heated to 95 °C and NSC in DMSO, or DMSO only, was added before snap-cooling on ice. RNA was then incubated for 1–2 h before native gel electrophoresis and densitometry analysis. a Dimerisation levels relative to the untreated RNA. Data are an average of five independent experiments, containing 1–3 samples of untreated/treated RNA each. Error bars show SD. b Representative samples from RNA renatured in the absence or presence of NSC, with (b) 2 h incubation at 37 °C or (c) 1 h incubation at 37 °C

Fig. 5

Structural effects of NSC on the gRNA. The first 411 nts of the gRNA, containing the packaging signal region and beginning of the gag ORF were in vitro transcribed and refolded. RNA was treated with NSC in DMSO or DMSO only. Acylation sensitivity at each nucleotide was used to model the structure and predicted free energy. The most stable structure is shown. a Predicted RNA structure and stability of DMSO treated RNA. b Predicted RNA structure and stability of NSC treated RNA. c, d Acylation sensitivity difference upon NSC treatment. c Differences > or < 0 and d differences > or < 0.1 reactivity units

Fig. 6

Schematic diagram showing the RNA packaging impairment upon NSC treatment. NSC may also be present inside virions