Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Abstract

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Keywords: Akt; Venezuelan equine encephalitis virus; antiviral; resveratrol.

Figure 1

50% cytotoxic dose and effective dose (CC50 and EC50) of resveratrol against Venezuelan equine encephalitis virus (VEEV) in two distinct cell types. Vero (A,B) and U87MG (C,D) cells were treated with resveratrol at indicated concentrations (CC50) along with VEEV-TC-83 infection for 16 h at an MOI of 0.1 followed by measurement (EC50) of (A,D) cell viability and (B,E) replication of reporter VEEV-TC83luc quantified by cellular ATP and luciferase activity, respectively. The figures were plotted following the normalizing toward viral control groups. Viral control refers to no resveratrol-treated but VEEV-infected cells, while mock control was defined as cells treated with a solvent and without infection. (E) The CC50, EC50, and therapeutic index (TI) were estimated via non-linear regression with GraphPad Prism v7.0 to determine the 50% cell viability and effective dose. Data were presented as means ± SEM.

Figure 2

VEEV replication and propagation of infectious virions were assessed after resveratrol treatment. Supernatants from VEEV-TC-83-infected Vero cells (MOI = 0.1) were collected at (A) 16 hours post-infection (h.p.i.) with various concentrations of resveratrol and (B) at the indicated time points with 125 μM of resveratrol, followed by performing plaque assays to measure viral titers. (C) Total cellular RNA was isolated from infected cells after 16 h of VEEV-TC83 infection (MOI of 0.1) followed by conducting RT-qPCR to quantify the viral RNA. (D) GFP-encoded VEEV-TC83 was used to quantify and visualize the VEEV-infected cells at 16 h.p.i. by (D) flow cytometry and (E) fluorescent microscopy. MFI, median of fluorescence intensity; VC, viral control. Data were expressed as means ± S.E.M. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to viral control (VC) by one-way ANOVA.

Figure 3

Time of addition of resveratrol marginally changed is antiviral effectiveness. (A,C) Schematic diagrams illustrating the experimental design for the time-of-addition experiments. (B) Luciferase activity and (D) viral RNA of VEEV-TC83luc at an MOI of 0.1 corresponding to the experimental designs were determined by BrightGlo^® assays and qPCR, respectively, at 16 h.p.i. (E) Apoptotic signaling activity of caspase-3 and caspase-7 in VEEV-infected Vero cells (MOI = 0.1) were measured with Promega’s Caspase-3/7-Glo^® assays at 16 h.p.i. *** p < 0.001; **** p < 0.0001 by one-way ANOVA. Data are represented as means ± S.E.M. VC, viral control.

Figure 4

Interaction between resveratrol and VEEV envelope glycoproteins. (A) Overview of VEEV E1 and E2 proteins, indicating the viewing angles corresponding to the following panel (B–D). Red arrows indicate the positions of resveratrol, and E1 and E2 are labeled in indigo and gold, respectively. (E) Viral attachment and entry were determined by quantifying the VEEV RNA inside cells at 16 h.p.i. in the presence or absence of resveratrol. ** p < 0.01 compared to viral control (VC) by one-way ANOVA.

Figure 5

The AKT pathway in VEEV-infected cells after resveratrol treatment. (A) Indicated protein level changes in AKT signaling pathways tested at 1, 3, and 6 h.p.i. by Western blot analysis with or without 125 μM of resveratrol along with VEEV-TC-83 infection at an MOI of 0.1. (B) The phosphorylation levels of AKT in VEEV-infected cells at 1 h.p.i. after resveratrol treatment at various concentrations and (C) the band intensities of AKT, GSK-3α, and GSK-3β were quantified with Quantity One analysis software from Bio-Rad Inc. (D) The fluorescent distributions of VEEV glycoprotein (GP) in green and cellular total AKT proteins in red were visualized in mock or VEEV-infected cells at an MOI of 0.5 and 4 h.p.i.

Figure 6

The broad-spectrum antiviral activity exerted by resveratrol. Viral titers of Sindbis virus (SINV), chikungunya virus (CHIKV), Rift valley fever virus (RVFV), and Zika virus (ZIKV) were evaluated at 24 h.p.i. after resveratrol treatment at concentrations from 0 to 200 μM. ND, undetectable. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to no-resveratrol-treated groups by one-way ANOVA.

Figure 7

EC50 and CC50 of resveratrol derivatives against VEEV. Pterostilbene (panel A) and piceatannol (panel B) were used to treat VEEV-TC-83luc (MOI = 0.1)-infected Vero cells at indicated concentrations, followed by measurement of EC50 by viral luciferase activity at 16 h post-infection (C,D). CC50 values were determined (E,F) via quantifying cellular ATP levels. The figures were plotted following the normalizing toward tissue or viral control groups.