Geneticin shows selective antiviral activity against SARS-CoV-2 by interfering with programmed -1 ribosomal frameshifting

Abstract

SARS-CoV-2 is currently causing an unprecedented pandemic. While vaccines are massively deployed, we still lack effective large-scale antiviral therapies. In the quest for antivirals targeting conserved structures, we focused on molecules able to bind viral RNA secondary structures. Aminoglycosides are a class of antibiotics known to interact with the ribosomal RNA of both prokaryotes and eukaryotes and have previously been shown to exert antiviral activities by interacting with viral RNA. Here we show that the aminoglycoside geneticin is endowed with antiviral activity against all tested variants of SARS-CoV-2, in different cell lines and in a respiratory tissue model at non-toxic concentrations. The mechanism of action is an early inhibition of RNA replication and protein expression related to a decrease in the efficiency of the -1 programmed ribosomal frameshift (PRF) signal of SARS-CoV-2. Using in silico modelling, we have identified a potential binding site of geneticin in the pseudoknot of frameshift RNA motif. Moreover, we have selected, through virtual screening, additional RNA binding compounds, interacting with the same site with increased potency.

Figure 1.

The activity of geneticin in maintained in Calu3 cells and in human-derived tissues. A) Calu3 cells were infected with SARS-CoV-2 (alpha variant) for 1 hour at 37°C. After the removal of the inoculum, the cells were treated with serial dilutions of geneticin. At 24 hpi supernatant was collected and viral RNA copies were evaluated with qPCR. B) and C) Mucilair tissues were infected with B) SARS-CoV-2 B.1.1 10⁶ RNA copies or C) SARS-CoV-2 Omicron BA.1 10⁵ RNA copies, the following day the apical treatment with 30 μg/tissue started. Every 24 hours an apical wash was performed and collected after 20 minutes at 37°C. The supernatant was then used for viral RNA quantification (solid lines) or for plaque assay (dashed lines). The results are the mean and SEM of two to three independent experiments performed in duplicate. P values <0.0332 (*), <0.0021 (**), <0.0002 (***), < 0.0001 (****)

Figure 2.

A) The EC₅₀s of geneticin were evaluated against the B.1.1.7 stock, viruses are grown in Vero E6 without treatment for 11 passages, or in presence of increasing doses of geneticin. B) The mutations observed at passage 11 as compared to the original B.1.1.7 stock (created with biorender.com).

Figure 3.

Mechanism of action of geneticin. A) Vero-E6 cells were infected with B.1.1.7 SARS-CoV-2 at MOI 0.1 and at MOI 0.01 (24hpi and 48hpi conditions respectively). Cells were treated post-infection with geneticin (600 μM). Cells were lysed 24hpi or 48hpi and protein quantification was done by Western Blot (Supplementary Figure 6). Values are expressed by the ratio of the intensity of SARS-CoV-2 nucleocapsid over alpha tubulin quantified by ImageJ. B) Vero-E6 were infected with SARS-CoV-2 at MOI 0.1 for 1 hour at 37°C. After the removal of the inoculum, geneticin (600 μM) or merafloxacin (100 μM) were added to the well. At 0, 4, 8 and 24 hours post.-infection cells were lysed and viral RNA was quantified. C) Dual luciferase evaluation was performed at 24 hours post-transfection in Vero-E6 cells treated with geneticin (600 μM) or merafloxacin (50 μM). The results are mean and SEM of three independent experiments performed in duplicate. P values <0.0332 (*), <0.0021 (**), <0.0002 (***), < 0.0001 (****). D) SARS-CoV-2 RNA frameshift-stimulatory element sequence and the mutant sequences.

Figure 4.

Comparison of the cryo-EM RNA structure (A) and the refined RNA structure by molecular dynamic simulation (B). (C) Different sequential mutations in the FSE structure: point mutations in Loop1 (in red); deletion of loop1 (in green); deletion of A and ACA in J3/2 (turquoise and yellow respectively). (D) The 3 binding sites identified by RNAsite. The binding site 1 (ring site); 2 (J3/2) and 3 (stem 2) are highlighted in red, blue and green, respectively. The 2D figures show geneticin interactions with the surrounding nucleotides in the binding sites. Turquoise dashed lines indicate weak H-bond; green dashed lines indicate strong H-bond, and purple dashed lines indicate electrostatic interactions

Figure 5.

Antiviral activity of site 1 −1 PRF binders against SARS-CoV-2. A) Table of analogs with increased potency against B.1.1.7 SARS-CoV-2 than geneticin. EC₅₀: half-maximal effective concentration, CC₅₀: half-maximal cytotoxic concentration. B,C,D) Mechanism of action of AB-3285. B) Vero-E6 were infected with SARS-CoV-2 at MOI 0.1 for 1 hour at 37°C. After the removal of the inoculum, AB-3285 (250 μM) was added to the well. At 0, 4, 8 and 24 hours post.-infection cells were lysed and viral RNA was quantified. C) Dual luciferase evaluation was performed at 24 hours post-transfection in Vero-E6 cells treated with AB-3285 (500 μM). The frameshift efficiency was normalized compared to untreated. D) Binding pose of AB-3285 in the PRF binding site 1. The results are mean and SEM of at least two independent experiments performed in duplicate. P values <0.0332 (*), <0.0021 (**), <0.0002 (***), < 0.0001 (****)