ABSTRACT
In this study, hesperetin was shown to inhibit the replication of multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus, and lumpy skin disease virus (LSDV). Hesperetin mainly suppressed viral protein synthesis without affecting other steps of the viral life cycle such as attachment, entry, and budding. In a chromatin immunoprecipitation (CHIP) assay, we further demonstrated that hesperetin-induced reduction in BPXV protein synthesis is due to disruption of the binding of the 5’ cap of viral mRNA with the cellular translation initiation factor eIF4E. The molecular docking and MD simulation studies, also confirmed binding of the hesperetin with the cap-binding pocket of eIF4E, in a similar conformation as m7GTP binds. In a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane, as well as the associated mortality of the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we for the first time demonstrated the antiviral activity of hesperetin against poxviruses, besides providing novel mechanistic insights into the antiviral action of hesperetin.
Keywords: Hesperetin, poxvirus, buffalopox virus, BPXV, eIF4E, m7GTP
INTRODUCTION
Poxviruses are a large family of DNA viruses that are capable of infecting a wide variety of animals including humans. While smallpox has been eradicated, the monkeypox virus has recently become an international concern for human health1. Likewise, the lumpy skin disease virus (LSDV) has emerged as the most important pathogen with regards to animal health 2. The poxviruses are usually host-specific 3. However, for reasons unknown, they may sometimes break the host tropism to infect unnatural hosts. For example, camelpox virus (CMLV) 4, buffalopox virus (BPXV) 5,6 and monkeypox virus (MPV)7 have zoonotic implications.
Some poxvirus inhibitors have been described 8-12. Among these, cidofovir is licenced to treat a variety of DNA viruses13 whereas Tecovirimat (previously known as ST-246) is the only FDA approved drug that specifically acts against orthopoxviruses 14. In addition, Brincidofovir, a prodrug of cidofovir, was approved in the United States in 2021 for the treatment of poxviruses. Besides having potential carcinogenic effects15, these directly virus-acting drugs are prone to induce drug-resistant mutants 16.
We screened a library of small molecule chemical inhibitors targeting host cell’s kinases and phosphatases and identified potential candidates with antiviral activity against BPXV. Hesperetin was identified as one of the inhibitors that blocked BPXV replication. Hesperetin (C16H14O6) is the aglycone form of the flavanone glycoside hesperidin (C28H34O15). It is a naturally occurring flavonoid found in citrus fruits such as oranges, grapes, and lemons and possesses anti-oxidant and anti-inflammatory properties by regulating various host signalling pathways17-19. Recent studies have also demonstrated the antiviral activity of hesperetin against some viral infections such as the chikungunya virus (CHIKV), Zika virus, Sindbis virus and dengue virus (DENV) 20-22. In this study we extended the antiviral efficacy of hesperetin against poxviruses, besides providing novel mechanistic insights on its antiviral action.
MATERIALS AND METHODS
Cells and viruses
African green monkey kidney (Vero) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with antibiotics and 10% foetal calf serum. Vero cell adapted BPXV (Accession Number VTCC-AVA90) and LSDV (Accession Number VTCC-AVA288) were available at NCVTC Hisar. Vaccinia virus (VV) was procured from the American Type Culture Collection (ATCC). BPXV and VV were quantified by plaque assay in Vero cells and LSDV was quantified by determination of tissue culture infective dose 50/ml (TCID50/ml) 23.
Inhibitors
Hesperetin was procured from Cayman Chemical (Ann Arbor, Michigan, USA). FR180204, CGP57380 and 4EGI-1 were procured from Sigma (Steinheim, Germany). The subcytotoxic concentration of ERK inhibitor (FR180204), MNK1 inhibitor (CGP57380) and eIF4E inhibitor (4EGI-1) were 0.2 μg/ml, 0.5 μg/ml and 0.5 μg/ml, respectively, and have been described previously by our group 24.
Antibodies
Anti-BPXV hyperimmune serum produced in rabbits was available at NCVTC, Hisar and has been described before 25. eIF4E monoclonal antibody (5D11) was procured from Invitrogen (South San Francisco, CA, USA). Anti-β-actin, Anti-Mouse IgG – alkaline phosphatase antibody, and anti-rabbit IgG – peroxidase antibody were received from Sigma-Aldrich (St. Louis, USA).
Cytotoxicity and virucidal activity
The cytotoxicity of hesperetin in Vero cells and virucidal activity against BPXV were determined as described before 26. Unless otherwise specifically stated, a non-cytotoxic concentration of 12 µg/ml of hesperetin or 0.05% DMSO (vehicle control) was used throughout the manuscript.
Time-of-addition assay
Confluent monolayers of Vero cells, in triplicates, were infected with BPXV at 5 MOI, followed by addition of hesperetin or DMSO (vehicle control) at -0.5 hpi, 1 hpi, 6 hpi, 12 hpi, 18 hpi, 24 hpi, 30 hpi and 36 hpi. Supernatants from the infected cells were collected at 48 hpi and quantified by plaque assay.
Attachment assay
Confluent monolayers of Vero cells, in triplicates, were treated with hesperetin or vehicle control for 1 h, followed by BPXV infection at 5 MOI for 1 h at 4°C. Cells were washed with PBS for five times to remove unattached virus, and cell lysates were prepared by rapid freeze-thaw cycles. The viral titres in cell lysates were quantified by plaque assay.
Entry assay
Confluent monolayers of Vero cells were infected with BPXV at 5 MOI for 1 h at 4°C to permit attachment. After that, cells were washed with PBS, and serum-free DMEM containing hesperetin or DMSO was added. This was followed by incubation at 37°C for 1 h which allowed virus entry. Thereafter, cells were washed again with PBS to remove any extracellular viruses and supplemented with DMEM without any inhibitors. The infectious virus particles released in the supernatant at 48 hpi were titrated by plaque assay.
Budding assay
Vero cells, in triplicates, were infected with BPXV at 5 MOI or mock infected for 1 h, followed by washing with PBS and incubation at 37°C. At 36 hpi, when BPXV presumably starts releasing from the infected cells, hesperetin or DMSO were added, and supernatants were harvested at 30 min and 4 h following the addition of the drug. Virus releases in the supernatants was quantified by plaque assay.
Viral protein synthesis
Vero cells were infected with BPXV at 5 MOI for 1 h. At 3 hpi, hesperetin or DMSO were added, and cells were incubated at 37°C. The cells were scrapped at 24 hpi and subjected to Western blot analysis by using hyperimmune serum raised against BPXV in rabbits.
qRT-PCR
The levels of viral DNA in the infected cells were measured by quantitative real-time PCR (qRT-PCR). Briefly, Vero cells, in triplicates, were infected with BPXV at an MOI of 5 for 1 h, followed by washing with PBS and the addition of fresh DMEM. Hesperetin or DMSO were applied at 3 hpi. Cells were scraped at 30 hpi to quantify BPXV C18L and the house-keeping gene (β-actin) gene as described before26.
In ovo antiviral efficacy
Specific pathogen-free (SPF) embryonated chicken eggs were procured from Indovax Pvt Ltd, Hisar, India. To determine the LD50, 4-fold serial dilutions of hesperetin (concentration ranging from 4000-62.5 µg/egg) or DMSO were inoculated in triplicates via the chorio-allantoic membrane (CAM) route in 10 day old embryonated SPF chicken eggs. The viability of the eggs was examined up to 5 days post inoculation and LD50 was determined by the Reed-Muench method 27.
For determination of the EC50, five-fold serial dilutions of hesperetin or DMSO, in triplicates were inoculated in 10 days old SPF embryonated eggs via CAM route, followed by infection of BPXV at 100 EID50. The eggs were examined for 6 days post-infection and CAM were harvested for examination of pock lesions. EC50 was determined by Reed-Muench method27.
Chromatin immunoprecipitation (CHIP) assay
The interaction between BPXV mRNA and eIF4E was performed using the CHIP assay as described previously by our group 28. Briefly, confluent monolayers of Vero cells in triplicates, were infected with BPXV at 5 MOI for 1 h, followed by washing with PBS. Cells were supplemented with fresh DMEM, and hesperetin or DMSO was added at 3 hpi. At 16 hpi, cells were treated with 1% formaldehyde to covalently cross-link the interacting proteins and nucleic acid for 10 min. The crosslinking was stopped using 125 mM glycine, followed by washing with ice-cold PBS. The cell lysates were prepared in immunoprecipitation buffer and sonicated as described before 28. The sonicated lysates were centrifuged at 12000 g for 10 min and clarified supernatants were mixed with 10 units of RiboLock RNase Inhibitor (Thermo Scientific, USA), followed by incubation with α-eIF4E (reactive antibody), α-MNK1 (nonreactive antibody), or equivalent volume of IP buffer (beads control) at room temperature. After 45 min, 40 μL (5 ng/μL) of Protein A Sepharose® slurry was incubated with each reaction at 4°C on a rotary platform overnight. The beads were then washed by IP buffer and crosslinking was reversed by addition of 20 mg/ml Proteinase K (followed by incubation at 56 °C for 40 min). The reaction mixture was then centrifuged at 12000 g for 1 min. The supernatant was subjected to RNA isolation, cDNA preparation, and quantitation of the BPXV Mgene by qRT-PCR.
Preparation of eIF4E for molecular docking and MD simulation
The crystal structures of eIF4E in open (PDB ID – 3TF2) and closed conformation (PDB ID - 1IPC) bound with m7GTP were used for protein visualization, docking studies and molecular dynamics simulation. Prior to analysis, the m7GTP ligand and water molecules were removed, and missing residues in the loop regions of crystal structure were modelled using SWISS-model 29. The structures were further energy minimised using AMBERff14SB force field 30followed by the addition of hydrogen atoms and water molecules as described previously 31. The prepared structures were used for subsequent docking and MD simulation studies.
Molecular docking, protein visualization and Ligplot analysis
The three-dimensional structure of hesperetin was obtained from Pubchem (Pubchem CID - 72281) while the m7GTP structure was taken from the PDB ID - 1IPC. The ligands were energy minimized and subjected to molecular docking using Autodock Vina 32 with eIF4E. First, a grid box was generated around the protein with centre coordinates; X= 46.5, Y= 106 and Z= -16 with the dimensions of the grid box; X= 44.5 Å, Y= 56 Å and Z= 54 Å. Thereafter, global ligand binding searches were performed. The result comprised nine best binding poses for ligands. For further evaluation, the best binding mode for each ligand was selected based on the binding affinity (Kcal/mol), RMSD lower- and upper bond. The protein and generated docked complexes were visualised using Pymol 0.99rc6. To assess the interaction of hesperetin and m7GTP with the eIF4E, Ligplot application was used 33. The docked complexes that resulted from Autodock Vina were saved in PDB format and uploaded to the Ligplot tool and the intermolecular interactions between the protein and ligands, such as polar and non-polar contacts were obtained in 2D representations.
Molecular dynamics (MD) simulation
To evaluate the stability and protein motion within internal coordinates, MD simulations of complexes with the highest affinities among the ligand and eIF4E were performed through the iMODS webserver (http://imods.chaconlab.org/) 34. The internal coordinates of the hespetin-eIF4E docked complex, the m7GTP-eIF4E docked complex, and the apo-eIF4E were saved in PDB format and further subjected to MD simulation through the iMODS webserver using normal mode analysis (NMA) for the calculation of the B-factor, structural deformability and eigen values, covariance mapping, and elastic network.
Selection of hesperetin-resistant BPXV mutants
BPXV was sequentially passaged (upto P=40) in Vero cells in the presence of hesperetin or DMSO. For each passage, confluent monolayers of Vero cells were infected with BPXV at an MOI of 0.1 for 1 h, followed by 5 times washing with PBS and the addition of fresh DMEM supplemented with hesperetin (3 μg/ml) or 0.05% DMSO. Supernatants were harvested at ~96 hpi or after observing a cytopathic effect (CPE) in ≥50% cells. The virus infected cell culture supernatant was used for the next passage (P). A total of 40 such passages were carried out. At the end of P40, the fitness of BPXV-hesperetin-P40 and BPXV-DMSO-P40 viruses was evaluated again in the presence of hesperetin.
RESULTS
Antiviral activity of hesperetin against poxviruses
Before examining the antiviral activity, we first determined a sub-cytotoxic concentration of hesperetin. At a concentration of ≤25 µg/ml, hesperetin had no effect on cell viability (Fig. 1A).The higher concentrations of hesperetin were toxic to the Vero cells(Fig. 1A) . Therefore, a sub-cytotoxic concentration of 12 µg/ml was used in the subsequent experiments. The cytotoxic concentration 50 (CC50) of hesperetin was determined to be 146.8 µg/ml(Fig. 1A) .
To evaluate the antiviral effect, virus yield in hesperetin-treated and untreated cells was determined by plaque assay. As compared to the vehicle-control (DMSO), a sub-cytotoxic concentration of 12 µg/ml resulted in reduced BPXV (Fig. 1B) , vaccinia virus (VACV)(Fig. 1C) and LSDV (Fig. 1D) yield. Further, preincubation of the extracellular virus (BPXV) with hesperetin had no effect on the viral infectivity, which suggested that the antiviral effect of hesperetin is due to inhibition of virus replication in the target cells and not simply due to inactivation of extracellular virions(Fig. 1E) .
Hesperetin suppresses BPXV replication at post-entry steps
To narrow down the specific time-points of the BPXV life cycle during which hesperetin may act to inhibit virus replication, we performed a time-of-addition assay where hesperetin was added at various times post infection (-0.5 hpi to 36 hpi) and supernatant was collected at 48 hpi for determination of virus yield. The levels of inhibition in virus yields were comparable, when hesperetin was added to the cells at -0.5 hpi, 1 hpi, 6 hpi or 12 hpi, suggesting that hesperetin may not affect the early stages (attachment, entry) of BPXV replication cycle(Fig. 2) . Likewise, virus yields were comparable in cells treated at 30 hpi or 36 hpi with hesperetin, suggesting that hesperetin is unlikely to affect the late stages (budding/release) of the BPXV replication cycle. However, the magnitude of viral inhibition was shown to progressively decrease from 18 hpi to 30 hpi of hesperetin addition, suggesting that hesperetin may act in the middle to pre-budding steps of the virus replication cycle (Fig. 2) .
Hesperetin has no effect on BPXV attachment, entry and budding
The effect of hesperetin on BPXV attachment to the host cells was evaluated by infecting the Vero cells at 4℃, which allowed the attachment of the virus to the cells but restricted viral entry. As shown in Fig. 3A, viral titres were comparable in both vehicle control-treated and hesperetin-treated cells, suggesting that hesperetin does not affect BPXV attachment to the host cells. To evaluate the effect of hesperetin on BPXV entry, the virus was first allowed to attach at 4oC in the absence of the drug, followed by incubating the cells at 37oC for 1 h (to allow viral entry) in the presence of hesperetin or vehicle control. As shown inFig. 3B, viral titres were comparable in both DMSO-treated and drug-treated cells, suggesting that hesperetin does not affect BPXV entry into the host cells. To evaluate the effect of hesperetin on virus release, hesperetin was applied when the virus presumably starts budding/release from the infected cells viz; at 36 hpi. As shown in Fig. 3C, viral titres were comparable in both DMSO- and drug-treated cells, suggesting that hesperetin does not affect BPXV egress from the infected cells.
Hesperetin treatment reduced the levels of viral DNA, mRNA and protein in the target cells
To evaluate the effect of hesperetin on the synthesis of viral DNA/mRNA/proteins, the drug was applied at 3 hpi, a time-point when the early steps of the BPXV life cycle (attachment and entry) have occurred. The cells were scraped at 24 hpi to quantify viral DNA/RNA/proteins. As shown in Fig. 4A, hesperetin treated cells showed ~72% reduction in viral mRNA copy numbers as compared to the DMSO-treated cells. Likewise, as compared to the vehicle-control treated cells, hesperetin-treated cells had ~26 %DNA (Fig. 4B). Since reduced levels of viral mRNA may reflect a reduction in the synthesis of viral proteins, levels of viral protein in drug-treated and DMSO-treated cells were also examined. As shown inFig. 4C (upper panel), hesperetin treatment remarkably suppressed the synthesis of viral proteins, whereas levels of the housekeeping control protein β-actin were unaffected (Fig. 4C, lower panel).
MAPK/ERK/eIF4E signaling axis is a prerequisite for BPXV replication
Viral mRNA translation in most DNA viruses (including poxviruses) and some RNA viruses occurs in a cap-dependent manner where eIF4E/eIF4G interaction plays a central role in translation initiation35. In agreement with the previous findings35, inhibition of the phosphorylation (activation) of ERK, MNK1 and eIF4E by small molecule chemical inhibitors (FR180204, CGP5738 and 4EGI-1, respectively) resulted in reduced BPXV replication(Fig. 5) suggesting the ERK/MNK1/eIF4E signalling axis is prerequisite for translation of BPXV proteins.
Hesperetin disrupts the interaction of viral mRNA with eIF4E
Further, the mechanism underlying the suppression of BPXV protein synthesis by hesperetin was elucidated. We performed a CHIP assay where cell lysates from BPXV-infected and, hesperetin-treated or untreated cells were immunoprecipitated by α-eIF4E and the viral mRNA in the immunoprecipitate was quantified by qRT-PCR. As shown in Fig. 6, as compared to DMSO-treated cells, the level of viral mRNA immunoprecipitated by α-eIF4E was significantly low. Further, the nonreactive antibody (α-MNK1) did not immunoprecipitate any viral mRNA. This suggested that e-IF4E specifically reacted with the 5’ cap of viral mRNA and that hesperetin blocks the binding of eIF4E and the 5’ cap of the viral mRNA.
Molecular docking and molecular dynamic (MD) simulations
In order to further confirm the binding of the hesperetin (Fig. 7A) at the 5’ cap (m7GTP, Fig. 7B)- binding pocket of the eIF4E, we performed protein visualisation, molecular dynamic (MD) simulations, and molecular docking studies (Fig. 7 and Fig. 8).We first superimposed the crystal structures of the open (Fig 7C, grey) and close (Fig 7C, green ) conformations of eIF4E using PDB id 3TF2 and 1IPC, respectively. A significant displacement in the loop region (49KNDKSKTWQANL60) of eIF4E in its mRNA cap-binding pocket was observed (Fig. 7C). In agreement with the previous reports 36,37, this flexible loop region adopted a closed conformation with an inward movement of ~6.4 Å (Fig. 7C zoomed view). This inward movement of the 49KNDKSKTWQANL60loop region (shown in red) provided anchoring points for the mRNA m7GTP cap. Another loop region, 203ATKSGSTT211(shown in orange) critical for the binding of the second nucleotide of the m7GpppA complex also showed an inward shift towards the cap-binding pocket 36. To confirm these shifts of the loop regions of eIF4E, we performed molecular dynamics simulations using iMODS server in normal mode analysis (NMA). As shown in Fig. 8A (Upper panel), we found similar movements in both loop regions of eIF4E.
The molecular docking studies revealed that both m7GTP and hesperetin bind to the same pocket of eIF4E (Fig. 7D and Fig. 7E ) with binding energies of -8.1 and -7.8 Kcal/mol respectively. In the crystal structure of m7GTP bound eIF4E, the m7GTP is stacked between the Trp56 and Trp102 residues of eIF4E 36. Similar conformation was also observed in the docking studies of m7GTP with eIF4E (Fig. 7D zoomed view). Interestingly, hesperetin also adopted identical stacking conformation between these two residues of eIF4E via its benzopyrone moiety (Fig. 7E zoomed view).
Interaction and stability of the hesperetin and eIF4E complex
For a detailed analysis of all the interactions between hesperetin and m7GTP with eIF4E, we constructed Ligplots 33 of the docked structures (Fig. 7F and 7G ) (Hydrogen bonds are highlighted in a green dashed line with bond lengths indicated in angstrom. The hydrophobic interactions are highlighted in red dashed lines). The methyl group of guanosine moiety which is an important 5’cap modification of mRNA and critically regulates its fate, interacted with the Trp56 residue of eIF4E via hydrophobic interaction. Remarkably, the methyl group of hesperetin also interacted with eIF4E via its Arg157 and Lys162 residues through non-polar interactions (Fig. 7G).
Several polar contacts were also observed in both docked complexes. Most notably, the eIF4E residues Glu103, Arg112 and Asn155 interacted with the nitrogen and oxygen atoms of m7GTP through hydrogen bonds. Owing to the multiple oxygen atoms in its ring structures, hesperetin also interacted with these three residues of eIF4E via hydrogen bonds, besides multiple non-polar contacts.
Overall, m7GTP interacted with eIF4E via 8 polar contacts, and 42 hydrophobic contacts while hesperetin interacted via 5 polar contacts and 49 hydrophobic contacts (Table 1).