In vitro and silico studies of geraniin interfering with HSV-2 replication by targeting glycoprotein D

Abstract Residues ASN94 and GLN41 presented the highest frequency in molecular docking tests. The geraniin-glycoprotein D(gD) complexes was stable with RMSD(root mean square deviation)value less than 0.3 nm. The Molecular dynamic (MD) simulations revealed stable hydrogen bonds between gD and geraniin. Root mean square fluctuation (RMSF) values were less than 0.15 nm around the interface of geraniin-gD complex. In virucidal assays showed a much higher anti-HSV-2 inhibition activity of geraniin as compared to acyclovir(ACV).Human immunodeficiency virus transactivator (HIV-TAT) treatment significantly enhanced HSV-2 replication and lethal effect on HaCaT cells. The inhibitory rate of geraniin against HSV-2 coinfected with HIV-TAT was significantly decreased. The immunofluorescence results also revealed that HSV-2 gD expression presented a green fluorescence on HaCaT cells membranes and showed clear downregulation in geraniin-treated cells, but was expressed clearly on cell membranes under geraniin, HSV-2 and HIV-TAT cotreatment. The anti-apoptotic effect from geraniin persisted after 72 h, while the anti-apoptotic effect from geraniin diminished when HIV-TAT and geraniin were combined. Graphical Abstract


Introduction
HSV-2 is enveloped DNA-viruses, causing recurrent infections in genital tract (Zhu and Viejo-Borbolla 2021).In the US, 17% of people are seropositive for HSV-2 (Cole 2020).The widespread prevalence of HSV-2 has promoted the occurrence of HIV infection, especially the recent occurrence of HSV-2 infection (Desai and Kulkarni 2015).HSV-2 has a complicated life cycle, which requires multiple viral glycoproteins and involves multiple host receptors.HSV-2 gD simultaneously combines with HVEM and nectin-1, a step that is critical for successful HSV infection of cells (Connolly et al. 2021).The chronic prevalence of HSV-2 infection has led to the long-term use of standard treatments, resulting in an enhanced risk of resistance to existing HSV-2 antiviral drugs, that primarily focus on the HSV replication cycle (Jiang et al. 2016).Therefore, agents that aim at HSV-2 replication and block the attachment of gD to host cell receptors have become a promising approach to prevent viral infection at an early phase (Kukhanova et al. 2014).HIV-TAT is a regulatory viral protein produced during HIV transcription early in the process and is secreted by infected cells after virus entry (Ajasin and Eugenin 2020).HSV-2 first lytic infection occurs within mucosal epithelial cells (Chodkowski et al. 2018).Expression levels of tight junction proteins in epithelial cells can be affected by HIV proteins TAT and GP120, leading to enhanced permeability of endothelial cells due to HIV infection, ultimately facilitating the infection, replication and spread of HSV infection (Sufiawati and Tugizov 2014).Geraniin, a polyphenolic compound commonly extracted from Geranium Wilfordii Maxim, has been used extensively to exhibit various biological activities (He et al. 2022).Although a series of studies on the reduction of viral infectivity in geraniin have demonstrated its promising ability as a broad-spectrum antiviral agent (Haddad et al. 2020), its detailed antiviral mechanism of action still requires further investigation.
The aim of this study was to determine the binding capacity of geraniin at the HSV-2 gD binding site, to determine the stability of the geraniin-gD complex during 50 ns MD simulations, and to determine the potential of geraniin as an anti-HSV-2 antagonist.In addition, we compared the anti-HSV-2 activities of geraniin to that of ACV both in the presence and absence of HIV-TAT.

Results and discussion
Our results of the docking study were outlined in Supplementary material, Table S1.Upon removal of the original ligand, it was re-docked into the original binding pocket in the PDB file, and the RMSD value was calculated by comparing the re-docked conformation with the original ligand conformation (Ganser et al. 2018).It was observed that docking calculations conducted by Autodock Vina were well in accordance with the experimental results (RMSD = 1.944Å) indicating a higher capability of predicting the native binding mode.Docking of original ligand into the crystal structure conformation of HSV-2 gD accurately reproduced the conformation of this ligand in the orthosteric pocket; the residues ASN171, ASN94, THR116 and GLU117 formed extensive hydrogen bonds and van der Waals contacts with the residues GLN41, ILE40 and THR92, and formed carbon-hydrogen bonds with TYR93 of HSV-2 gD contributing to a docking affinity of −5.6 kcal/mol.Entry/fusion inhibitor docosanol was successfully docked in the same pocket than the natural ligand; the residues GLN41 and ASN94 formed extensive hydrogen bonds and van der Waals contacts with the residues HIS39, GLU117, THR116, ILE129, VAL37 and ILE40 of HSV-2 gD contributing to a docking affinity of −4.7 kcal/mol.Geraniin was shown to form complexes within the same binding pocket than original ligand on HSV-2 gD and formed H-bond with GLU45, GLN41, ASN171, THR92, THR116, ASN94 and ILE40 residue with optimal docking score of −7.4 kcal/mol.RMSD was then calculated on the optimal superimposition, the RMSD for geraniin conformation was 1.211 Å, which was similar to the original ligand (RMSD = 1.944Å) .
Similarly, the docking results using Autodock Vina showed that the selected ligands were also docked at the same binding site, in which all interactions between ligands and proteins concerned residue ASN94.Following Glide and Libdock docking analysis, geraniin consistently showed the best docking score (-5.462 and 114.927, respectively).Geraniin and N-Acetyl-D-Glucosamine(NAG) both bound to HSV-2 gD through several direct hydrogen bonds with a key residue, ASN94.As shown in Supplementary material, Figure S1, the docked conformation of Vina revealed that geraniin docked inside the binding regions of HSV-2 gD with GLU45 and GLN41.The docking results of Vina also suggested that GLN41 residue in HSV-2 gD formed hydrogen interactions with docosanol and van der Waals contacts with NAG.Following the docking results based on Glide, the GLN41 residue in HSV-2 gD formed a hydrogen bond with geraniin.Based on crystal structures of HSV-2 gD, we speculated that ASN94 and GLN41 residues might also be critical for HSV-2 gD interaction with selected ligands.These two residues were identified to form critical interactions in the reported crystal structure of HSV-2 gD as well (Lee et al. 2013).
The binding mode of geraniin at different simulation stages (0 ns and 50 ns) were showed in Figure S2.Within the 10 ns time frame, the geraniin-gD complex was found to exhibit larger conformational fluctuations.During 10 ns~30ns range, the conformers seeked a conformation that minimized the energy and tended to be stable.Upward trend increases in RMSD value had occurred after about 30 ns, but there was a tendency to smooth and stabilize.Throughout the dynamics simulation, the RMSD value varied little (amplitude less than 0.3 nm), demonstrating that the conformational stability of the model structure was achieved.
RMSF values were lower around the interface of the ligand-protein complex, indicative of reduced fluctuations and a more stable interaction.The backbone Cα RMSF values fluctuated little (magnitude smaller than 0.15 nm), indicating that the dynamics of the entire protein was conservative, which were conducive for the formation of stable geraniin-gD complex (Figure S3A).The RMSF values for each residue were calculated, and then colored each according to the RMSF value (Figure S3B).The most obvious characteristic of this plot was that there were noticeable areas with a high degree of flexibility around protein initiation site as opposed to other parts of the protein structure.The 'loop' regions of HSV-2 gD were located in the disordered region on the right side of Figure S3B, whereas proteins that interacted with geraniin were on the upper left side.Secondly, there were obvious fluctuations in the side chain locations of residue 65, which were located below the protein posterio and had no major impact on the general organization of the active site residues.The loops near residue 175 located underneath the protein fluctuated obviously and were far away from the active site for which had little effect on residues near the active site.
Hydrogen bond networks is crucial for understanding protein-ligand binding interactions (Schiebel et al. 2018).As shown in Figure S4, the overall network with an average of 6 hydrogen bonds remained stable throughout the simulation which indicated that geraniin-gD complex were always interacting via a hydrogen bond.
In order to better identify the phase of viral replication that was influenced by geraniin, the chemical was incorporated into the virus or into the cells before the infection or into the cells after the infection.As shown in Figure S5A, cell viability was significantly lower in the model group than in the control group.With different modes of medication, geraniin displayed significant prolongation of HaCaT cells survival rates (p < 0.001) and viral inhibition rates (p < 0.01) in the therapeutic and virucidal groups as compared to ACV-treated therapeutic group (Figure S5A,B).The antiviral effects of test compounds was assessed by quantifying HSV-2 gD and viral tegument protein 16(VP16) mRNA levels using real-time PCR.The gD and VP16 mRNA expressions levels were significantly increased after virus infection (Figure S5C,D).The highand middle-dose of geraniin groups displayed markedly higher viral inhibition effect towards gD and VP16 mRNA expressed levels than in the model group (p < 0.0001).After 72 h, the cell cytotoxicity of HIV-TAT proteins was tested with MTT assay and no significant cytotoxicity was observed at concentrations tested.HIV-TAT protein could act directly on HaCaT cells to enhance cell Membrane permeability (Sufiawati and Tugizov 2014), not causing cell death at concentrations tested (Figure S5E).We also investigated the impact of HIV-TAT towards HSV-2 replication.The survival rate of the HSV-2 infected group (40.3%) was considerably smaller than in the control group (Figure S5F).During HSV-2 infection of HaCaT cells, the HIV-TAT protein promoted the HSV-2-induced cell mortality rate.At concentrations ≥6.25 ng/ml of HIV-TAT protein, HSV-2 and HIV-TAT cotreatment resulted in decreased cell survival rates compared with the HSV-2-alone groups (p < 0.05).In the presence of 25 ng/mL of HIV-TAT proteins, the 3-day survival rate was 34.4% for HSV-2 and HIV-TAT cotreatment group as opposed to the control group.Therefore, this concentration of HIV-TAT was selected as the basis for the follow-up assays.We also investigated the effect of HIV-TAT with treatment as well as with HSV-2 infection on the antiviral activity of selected compounds.Although HIV-TAT had facilitated HSV-2 replication (Horbul et al. 2011), it does not appear to interfere with the cytoprotective and viral inhibitory ability of ACV to block HSV-2 (Figure S5G,H).As shown in Figure S5G,H, cell survival rates and viral inhibition rate were markedly decreased in the high-dose geraniin + HIV-TAT-treated group, compared to the HSV-2-alone group.Antiviral therapy with ACV inhibited the viral DNA polymerase but not viral fusion, so it remained active under HSV-2 and HIV-TAT cotreatment.Thus, we speculated that geraniin might directly affect the adhesion to the entry receptor or the fusion of the virus to the host membrane by targeting the gD protein.
As geraniin might inhibit HSV-2 binding by affecting the conformation of gD, the ability of geraniin to block gD-mediated membrane fusion was assessed in immunofluorescence assays.As shown in Figure S6, control HaCaT cells without HSV-2 treatment displayed normal cell morphology with no expressing HSV-2 gD tagged with green fluorescent protein.Cells in the HSV-2-alone group were unevenly green colored with fractured morphology.And then, as shown in ACV and geraniin treatment groups without HIV-TAT treatment, numerous normal HaCaT cells were stained blue with clear and intact nuclei in contrast to the HSV-2-alone group.Furthermore, both the intensity of blue fluorescence and the number of cells decreased markedly, whereas the HSV-2 gD expression(green) increased in the HIV-TAT and HSV-2 combined-treatment group in contrast to HSV-2-alone group.We observed that HIV-TAT increased the production of potentiated green fluorescent protein and reduced the number of normal cells when HIV-TAT, HSV-2 and graniin were combined compared to HSV-2 and graniin combined group, suggesting that HIV-TAT prevented geraniin inhibitory effect on HSV-2.
Apoptotic analysis showed that the rate of apoptotic cells was about 10.9% in non-infected group (Figure S7A), while it was about 31.6% in infection group (Figure S7B), which suggested that HSV-2 infection could increase cell apoptosis.Cell apoptosis rate of HaCaT cells was significantly increased (41.4%, Figure S7C) with HSV-2 infection combined with HIV-TAT treatment.HSV-2-induced apoptosis is important for virus replication, and the activity apoptotic death of HaCaT cells was accelerated by HIV-TAT.The ACV-treated group with HSV-2 infection showed an extremely low apoptosis rate of 12.2% (Figure S7D).The apoptosis rate in geraniin-treated group was about 16.4% (Figure S7E), while reached to 19.3% when HIV TAT, HSV-2 and graniin were combined (Figure S7F).These results suggested that treatment with HIV-TAT protein attenuated anti-apoptotic effects of geraniin in HaCaT cells with HSV-2 infection.

Experimental
The materials and methods adopted for the study had been described in the Supplementary material.

Conclusions
From preventative and therapeutic viewpoints, targeting HSV-2 gD is a hopeful strategy towards the development of promising anti-HSV-2 drugs (Du et al. 2017).Our findings showed that geraniin might serve as a membrane fusion inhibitors for HSV-2 gD in which the binding affinity for geraniin was found higher than in NAG and docosanol.Our results from MTT, QPCR, Flow cytometry and immunofluorescence assays confirmed geraniin having the virus inhibition effect and Interference ability with gD-dependent membrane fusion, which was an essential step in the HSV-2 entry.Thus, these results presented experimental proof, uncovering the impact and some of the molecular mechanisms that geraniin might modulate HSV-2 infection by directly targeting HSV-2 gD.The geraniin was also found to be more effective than ACV in virucidal patterns against HSV-2 with higher viral inhibition activity.This offered many interesting possibility worthy of further research and discovery, and had a broad application prospect in genital herpes diseases.