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. 2017 May 24;7(1):2341.
doi: 10.1038/s41598-017-02667-3.

A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication

Sara Callegaro  1 , Rosalba Perrone  1 , Matteo Scalabrin  1 , Filippo Doria  2 , Giorgio Palù  1 , Sara N Richter  3
Affiliations

A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication

Sara Callegaro et al. Sci Rep. .

Abstract

G-quadruplexes (G4s) are nucleic acids secondary structures, epigenetic regulators in cells and viruses. In herpes simplex virus 1 (HSV-1)-infected cells, G4s are massively present during viral replication. We here aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphtalene diimide (c-exNDI) G4 ligand. Biophysical and biomolecular analysis proved that c-exNDI stabilized the HSV-1 G4s in a concentration dependent manner. In MS competition assays, c-exNDI preferentially recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells; other less abundant cellular G4s were also recognized. Treatment of HSV-1 infected cells with c-exNDI at low nanomolar concentrations induced significant virus inhibition with no cytotoxicity. The mechanism of action was ascribed to G4-mediated inhibition of viral DNA replication, with consequent impairment of viral genes transcription. Our data suggest that the observed potent antiviral activity and low cytotoxicity mainly depend on a combination of c-exNDI affinity for HSV-1 G4s and their massive presence during infection. HSV-1 G4s may thus represent new effective antiviral targets: the fact that no current antiherpetic drug exploits them and their presence at the viral genome, responsible for both active and latent HSV infections, makes them particularly attracting.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Thermal unfolding of un3 parallel G4 (a), gp054a hybrid G4 (b), un2 antiparallel G4 (c) (4 μM) in 100 mM K+ in the absence (left panel) or presence (right panel) of 16 μM c-exNDI. Thermal unfolding was recorded over a temperature range of 20–90 °C with temperature increase of 5 °C.
Figure 2
Figure 2
Taq polymerase stop assay on HSV-1 G4 folding sequences in the presence of c-exNDI. (a) un2, un3 and gp054a were analyzed in the absence (lanes 1) or presence of 10 mM K+ (lanes 2–5). c-exNDI was used at concentrations of 37.5, 75 and 100 nM (lanes 3–5). Elongation was performed at 60 °C. A non-G4 folding sequence was used as negative control (no G4 cnt). G4-related stops are indicated by vertical bars. FL stands for “full-length” amplified template. P indicates the primer lane. (b) Quantification of c-exNDI-induced stop sites observed in (a).
Figure 3
Figure 3
Anti-HSV-1 activity of c-exNDI. (a) Plaque assay: Vero cells were infected with HSV-1 strain F (MOI 1, as previously reported) and treated with increasing concentrations of c-exNDI (0.7 nM–100 nM). Supernatants were collected 24 h.p.i. and the number of plaque forming units was determined. (b) Vero cells were treated at the same concentrations of c-exNDI (0.7 nM–100 nM) used in the antiviral assay and cytotoxicity was evaluated by MTT assay. (c) Flow cytometry of HSV-1 [V41]-infected cells treated with c-exNDI and ACV. HSV-1 [V41]-infected cells were used as positive control and their GFP fluorescence set to 100%. C-exNDI (red curve) and ACV (orange curve)-treated infected cells were compared to HSV-1 [V41]-infected cells (green curve) to monitor compound inhibition of GFP fluorescence. (d) Quantification of the mean of GFP fluorescence upon treatment of HSV-1 [V41]-infected cells with c-exNDI and ACV, observed in (b).
Figure 4
Figure 4
Antiviral effects of c-exNDI. (a) Effect of c-exNDI on mRNA levels of immediate-early (IE), early (E) and late (L) proteins of HSV-1. Infected cells were treated with c-exNDI (100 nM); 4 or 24 h.p.i total RNA was isolated, retrotranscribed into cDNA and expression of specific genes was determined by RT-PCR. RQ are Relative Quantities. Each gene was analyzed in duplicate. In all data sets: n ≥ 2, mean ± s.d., Student’s t-test, p ≤ 0.01. (b) Effect of c-exNDI on HSV-1 cycle steps evaluated by time of addition assay. C-exNDI was tested at 100 nM (dashed line/squares), acyclovir (ACV) was used as a reference drug and tested at 3 μM (solid line/circles). Data from infected cells treated in the same conditions but without the compounds are reported as dashed line/triangles. Compounds were administered from 0 to 12 h.p.i. and supernatants were collected 30 h.p.i. The left and right Y-axis refer to ACV and c-exNDI data, respectively.
Figure 5
Figure 5
Scheme of the proposed c-exNDI mechanism of anti-HSV-1 activity.
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