Vaccinia virus virulence factor N1L is a novel promising target for antiviral therapeutic intervention

Abstract

The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified submicromolar (600 nM) N1L antagonists belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70-1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus.

Figure 1. Determination of IC₅₀ values of select N1L antagonists by fluorescence polarization experiments

A. Ligand 7; IC₅₀(N1L) = 0.6 μM; IC₅₀(F1L) = 1.4 μM. B. Ligand 11; IC₅₀(N1L) = 0.9 μM; IC₅₀(F1L) = 1.7 μM. C. Ligand 12; IC₅₀(N1L) = 0.9 μM; IC₅₀(F1L) = 0.6 μM; IC₅₀(Bcl-2) = 4.7 μM; IC₅₀(Bcl-XL) = 8.5 μM. FPA, normalized fluorescence polarization; Log([L], M), decimal logarithm of the ligand concentration in M. Data were fit to the variable slope dose-response equation. FPA assay was performed as described in Methods. Incomplete titration curves (ligand saturation was not achieved in a given concentration range) are shown for Bcl-2 and Bcl-XL in panels A and B solely to demonstrate poor ligand binding to these proteins.

Figure 2. Thermodynamics of ligands interaction with N1L as determined by differential scanning calorimetry

A (−), N1L, 71μM, DMSO only, T_m=58.6 °C, ΔH=13.1 kcal/mol, ligand 3: 214 μM, T_m= 54.4 °C, ΔH=11.0 kcal/mol; ligand 6: 71 μM, T_m= 57.5 °C, ΔH=8.7 kcal/mol; resveratrol: 214 μM, T_m= 54.2 °C, ΔH=10.3 kcal/mol. B. (−), DMSO only, T_m=57.2 °C, ΔH=16.1 kcal/mol, ligand 7: 106 μM, T_m= 54.9 °C, ΔH=12.3 kcal/mol; ligand 9: 106 μM, T_m= 52.3 °C, ΔH=17.8 kcal/mol; ligand 11: 106 μM, T_m= 56.3 °C, ΔH=20.3 kcal/mol; ligand 12: 106 μM, T_m= 48.6 °C, ΔH=11.2 kcal/mol. C. N1L, 71 μM protein, DMSO, T_m=58.6 °C, ΔH=13.1 kcal/mol; N1L-Resv, 71 μM protein, 214 μM resveratrol, T_m=54.2 °C, ΔH=10.3 kcal/mol; N1L I6K, 71 μM protein, DMSO, T_m=61.5 °C, ΔH=15.8 kcal/mol; N1L I6K – Resv, 71 μM protein, 214 μM resveratrol, T_m=61.1 °C, ΔH=15.9 kcal/mol.

Figure 3. Inhibition of vaccinia virus growth by N1L antagonists

A. VacV-GFP growth inhibition EC₅₀ values obtained in CV1 and HT1080 cell lines. B. Ligand cytotoxicity in the absence of VacV-GFP. The indicated ligands were added to the cells at 150 μM. The ligand numbering corresponds to that of the Table 1.