Abstract
Herpes virus1(HSV1) is a neurotropic virus that has been linked to Alzheimer’s disease. An In-silico structural homology search using α -Bgtx, identified structural homology between HSV1 gD and the nicotinic receptor neurotoxin α-Bgtx. SPR binding studies using acetylcholine binding protein from Lymnaea stagnalis, and functional two electrode voltage clamp studies of α7 nAChRs demonstrate the ability of HSV1 to interact directly with nAChRs. Molecular docking studies support the binding of a neurotoxin-like binding loop in HSV1 to a binding site similar to the neurotoxin binding domain. Interaction of HSV1 with nAChRs provides novel insights into a potential mechanism of action of HSV and its potential role in Alzheimer’s disease.
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Introduction
Rabies, herpes, and influenza viruses are neurotropic viruses known to produce neurological symptoms in infected individuals through direct neuronal damage or alterations in the host immune system pathways1. This study focuses on the interaction of herpes simplex virus 1 (HSV1) with nicotinic acetylcholine receptors as a novel potential mechanism of viral mimicry in altering the host response. Herpes viruses are classified into three groups: α-herpes viruses (herpes simplex virus types 1, 2), β-herpes viruses (human herpes viruses 6 and 7, and cytomegalovirus), and the γ-herpes viruses (human herpes virus 4 and 8)2,3. Several herpes viruses have known neurotropic effects, although the mechanisms of these effects are not well established. Among these are HSV1, HSV2, HHV3 (varicella-zoster), HHV4 (Epstein Barr), and HHV64.
Herpes simplex viruses are endemic. The α-virus, HSV1, known for its mouth infection, “herpes labialis”, affects over 40 million people in the USA with most infections occurring in childhood5. HSV1 primarily infects stratified squamous epithelium of oral and anogenital mucosa. The virus spreads into sensory neurons of the mucous membrane and then travels via retrograde axonal transport to the neuronal cell body6. In the trigeminal and dorsal root ganglia (DRG), HSV1 usually establishes a lifelong latent infection7. Reactivated HSV1 may enter the CNS where it can cause severe encephalitis8. Although several studies suggested that the latent herpes virus reactivates in the trigeminal ganglia (TG) and then travels to the CNS, one cannot eliminate the possibility of herpes virus reactivation in the CNS directly9. Studies using tissue samples from Alzheimer’s patients and normal individuals found a higher presence of HSV1 gD DNA in the frontal cortex and cerebellar regions of the brain. These studies suggested a possible correlation between HSV1 and AD10,11. HSV1-induced herpes simplex encephalitis (HSE) is a condition where the reactivated latent virus causes severe inflammation in the brain12. Patients with HSE experience a range of neurological symptoms including amnesia and reduced cognitive ability. Reactivated HSV1 virus has been found in the temporal lobe, frontal lobe, and hippocampus. Several studies are investigating HSV1 virus, and other herpes viruses, as possible contributors to the development of Alzheimer’s disease (AD)13.
Herpes virus 1 follows common herpes viral mechanisms of invading host cells by membrane fusion. Different glycoproteins of HSV1, including glycoproteins B, C, D, E, I, and H, play vital roles in spreading the virus from cell to cell14. Herpes virus glycoproteins initiate a complex membrane fusion process that starts with interacting with the virus glycoprotein and host cell receptors15. HSV1 has four essential glycoproteins responsible for host cell fusion: gB, gD, gL, and gH16,17. The interaction of gD with host receptors initiates the cascade of cell entry18. Among the HSV1 glycoproteins, gD has been established as the main determinant for cell recognition and entry for most α-herpes viruses19. Glycoprotein D is unique to HSV1 and HSV2 and does not occur in other herpes viruses, including the alpha herpes virus Varicella-zoster. The presence of the gD in HSV1 and 2 may play a role in their unique neurotropic effects. Interactions of gD in the CNS could mediate these effects.
Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and peripheral nervous systems but also play important roles in non-neuronal cells. α7 nAChRs are found on numerous immune cells, including T and B lymphocytes, macrophages, and mast cells, indicating a role of these receptors in modulating the immune response20. Inhibition of α7 receptors of the vagus nerve produces increased inflammatory responses. Modulation of nAChRs by viruses could enhance their ability to enter host cells, replicate within them, and evade the host immune response. This has been observed in viruses that express surface proteins structurally similar to CD59. CD59 is a member of the LY6 family proteins containing a conserved LU domain21. CD59 is a complement regulatory protein that protects cells from complement-mediated lysis by inhibiting the formation of membrane attack complex (MAC)22. This mimicry of endogenous proteins leads to dysregulation of the immune system23. Through viral mimicry, viruses can dampen the host immune response, allowing them to evade detection and clearance by the immune system24.
It has been noted in prior research that some viral proteins (e.g. rabies virus glycoprotein, HIV gp120, and SARS-Cov2) contain a loop region that is similar to a 3-fingered toxin loop (3FTx) region of α-neurotoxins such as α-bungarotoxin (α-Bgtx)25,26. This family of neurotoxins has been extensively characterized and found to inhibit nAChRs. The presence of a homologous sequence in some viruses suggests that they may also be able to interact with nicotinic receptors. In the rabies virus, this interaction appears to mediate the behavioral effects of the virus in the CNS27. In other viruses, the effect is not well known; however, there have been reports of using nicotine patches in treating long Covid, possibly suggesting a nicotinic mechanism28. The involvement of HSV1 and HSV2 in Alzheimer’s disease suggests a similar nicotinic receptor interaction may be possible for HSV. It is of note that the 3FTx family neurotoxins are highly homologous to several endogenous regulatory proteins that form the LY6 family including the nicotinic regulatory proteins LYNX, SLURP, and the membrane-bound complement regulatory protein CD59, suggesting that viruses may be mimicking these endogenous proteins to modulate host response29,30.
The studies described here investigate the potential binding of herpes viruses to nAChRs using a combination of structural and sequence homology studies, receptor binding, functional experiments, and in-silico docking. Our data indicates a high degree of structural homology between the binding loop region of the 3FTx protein α-Bgtx and a loop region of HSV1 gD. Subsequent binding and functional studies confirm an interaction between this HSV1 gD loop region and nAChRs. We have tested a whole ectodomain of HSV1gD, 92AA HSV 1gD fragment protein, and 24AA loop peptide region to examine our hypothesis that the presence of a structurally similar loop region of 24AA enables the virus to interact with α7 nAChRs. We also compared the potency of interaction between the whole ectodomain of HSV1gD, 92AA HSV 1gD fragment protein, and 24AA loop peptide region. We present docking studies that indicate a potential binding region for HSV1 gD near the α-Bgtx binding region of the nAChRs. Our data provide, for the first time, a potential mechanism connecting HSV1 to AD and, in combination with studies from Rabies and SARS-Cov2, may suggest that a common structural motif may be present in viruses that mediate neurotropic effects via nAChR interaction in the brain, potentially leading to long term neuropathology.
Results
Structural and sequence homology between HSV1 gD and α- Bgtx
Structural homology modeling can determine the level of structural similarity in proteins and domains that may or may not have significant sequence homology31. DNASTAR’s Lasergene, an integral Protean 3D application, was used for structural homology searches and analysis. The Protean 3D tool is used to predict protein’s secondary structures, identify functional domains, and perform a structure modeling32. Two alignment methods, TM-Alignment and jFATCAT were used for structural analysis to compare HSV1 glycoprotein D with α-bungarotoxin.
The structural homology level or the structural alignment quality was quantified using the global Root Mean Square Deviation (RMSD), a measure of the average distance between atoms in the peptide backbone when two molecules are superimposed. In general, an RMSD ≤ 0.5 Å is considered an indication of a near-identical structural similarity, an RMSD between 0.5 - 2.0 Å corresponds to a high degree of similarity, an RMSD between 2.0 - 5.0 Å indicates a lower, but still noticeable, degree of similarity.
Investigation of the structural homology between HSV1 gD (PDB ID: 2C36) and α-Bgtx (PDB ID: 1HC9) using TM-Alignment and jFATCAT revealed a similar 3FTx loop structure with RMSD values of 1.981 Å and 2.249 Å respectively. Figure 1a and b show the loop region of the HSV1 gD ectodomain (1a, G83 - P92, shown in blue) and the structurally homologous region in α- Bgtx (1b, C29-V40, shown in red). Figure 1c and d show the overlapping structures, illustrating the highly structurally homologous loop region (TM-Alignment: Fig. 1c, and jFATCAT alignment: Fig. 1d). A detailed analysis of the TM-Alignment (Fig. 1c, RMSD 1.981 Å) indicates a close alignment between F32, S34, and S35 of α-Bgtx (red) with V88, K90, and Q91 of HSV1 gD (blue). The side chains of these residues extend away from the loop in a position available for binding. A somewhat different alignment of amino acids was observed using the jFATCAT alignment model (Fig. 1d, RMSD 2.249 Å). In this model, Q91, D87, and K90 of HSV1 gD (blue) are aligned with R36, F32, and S35 of α-Bgtx (red), respectively, with side chains oriented in a position appropriate for binding.
a The nAChR 3FTx loop 2 binding region of α-Bgtx is shown in red. b A loop region in HSV1gD that displays significant structural homology to the 3FTX loop 2 region of α-Bgtx is shown in blue. c The homologous loop structures of α-Bgtx 3Ftx loop (red) and HSV1 gD (blue) are overlapped using the TM-align algorithm (RMSD = 1.981 Å). A close-up view of the model (right) depicts specific amino acids side chain alignments. d The homologous loop structures of α-Bgtx 3Ftx loop (red) and HSV1 gD (blue) are overlapped using the jFATCAT algorithm (RMSD = 2.249 Å). A close-up view of the model indicates the alignment of individual amino acid side chains.
While HSV1 gD and α-Bgtx show substantial structural homology, a sequence comparison of α-Bgtx (C29 – V39) and HSV1 gD (G83 – L95) using Clustal, Omega, ClustalW, MAFT, and MUSCLE algorithms failed to show a good sequence homology. Using both Clustal Omega and ClustalW, only a 28% sequence similarity was indicated.
Binding and functional studies of the HSV1 gD whole ectodomain with nicotinic receptors
The acetylcholine binding protein (AChBP) is a useful and well-characterized nAChR homolog used to analyze the extracellular domain of pentameric LGIC receptors33. The AChBP is a soluble, yet highly homologous, protein with sequence and substantial structural similarity to the extracellular domain of nAChRs. Due to its solubility, the AChBP is amenable to surface plasmon resonance (SPR) studies that can be used to determine the interactions of other molecules with the extracellular domain of nAChRs receptors34,35. SPR was used in this study to evaluate the direct interaction of HSV1 gD with the AChBP and, thus, its potential to interact with membrane-bound nAChRs. SPR studies provide both affinity (Kd) and binding rates (k1 = on rate and k2 = off rate) for interactions between the whole ectodomain of herpes1 gD and the receptor.
For SPR studies, L-AChBP was immobilized on the Carboxymethyl dextran (CM) coated chip surfaces as described in the methods. Clear binding of the whole ectodomain of HSV1 gD (319 AA, UniProt ID A0A1C3J7P6) to the L- AChBP was observed in these experiments with a Kd = 2.12 × 10-6 ± 5.31 × 10-7 M. The kinetics of binding are shown in Fig. 2a.
a HSV1 gD whole ectodomain binding to the L -AChBP. SPR was conducted using four different concentrations of HSV1 gD whole ectodomain (7.8 nM, 31 nM, 1.2 μM and 5 μM). L-AChBP protein was immobilized on the CM chip surface (the ligand) and HSV1 gD was flowed over the chip surface (the analyte). Analyte was injected for 4 min at a flow rate of 25 μL/min. Affinity was calculated using a 1:1 binding model (Kd = 2.12 × 10-6 ± 5.31 × 10-7 M). b Functional effects of HSV1 gD on α7 nAChRs expressed in Xenopus oocytes. Oocytes were injected with mRNA coding for the human α7 nAChR. Oocytes were then co-exposed to both nicotine (30 μM) and the 319 AA whole ectodomain of HSV1 gD. 30 μM nicotine produced approximately a 40% Imax response that was inhibited by increasing concentrations of HSV1 gD. The four different concentrations of HSV1 gD shown (0.1 μM, 0.33 μM, 1 μM, 2 μM) produced an area under the curve (total current) of 502, 331, 138, and 10 uA·ms respectively. c Dose response curve for inhibition of nicotinic induced responses by HSV1 gD. The AUC data obtained from oocyte inhibition experiments was used to create an inhibitory dose response curve for HSV1 gD. Data from at least 4 oocytes was pooled to create the curve shown. IC50 = 0.85 µM (95% CI: 0.55 to 1.14 µM).
The binding of HSV1 gD to the L-AChBP suggests that it may be able to bind to nAChRs. To characterize the interaction and functional effect of this binding, Xenopus oocytes expressing human α7 nAChRs were exposed to varied concentrations of the same HSV1 gD whole ectodomain that was used in the L-AChBP SPR experiments. Using a fixed nicotine concentration of 30 μM (equivalent to an EC40 concentration of nicotine) to elicit a response, oocytes were co-exposed to HSV1 gD whole ectodomain at concentrations ranging from 0.1 μM to 2 µM and the response relative to nicotine alone was determined (Fig. 2b). HSV1 gD inhibited the nicotine-induced response in a concentration-dependent manner with an IC50 = 0.85 µM (95% CI: 0.55–1.14 µM).
Binding and functional studies of HSV1 gD 92AA protein with nicotinic receptors
To determine if a smaller fragment of HSV1 gD ectodomain containing the putative binding loop region could still bind nAChRs, a fragment of HSV1 gD ectodomain containing the homologous loop region (92AA, PDB ID: 2C36 Y59-G150) was analyzed identical to the whole ectodomain as described above (Fig. 2). As shown in Fig. 3a, the shorter, 92 AA acid fragment also revealed an interaction with L-AChBP using SPR analysis, producing a Kd equivalent to the whole ectodomain (kd = 1.07 x 10-7 M, ± 1.32 x 10-8 M).
a Binding to the L–AChBP, SPR binding data study showing binding at four different concentrations (10 nM, 40 nM, 200 nM, 1 μM) of the 92AA HSV1 gD protein. Analyte (92 AA fragment) was injected for 4 min at a flow rate of 25 μL/min. Affinity was calculated using a 1:1 binding model (Kd = 1.07 × 10-7 ± 1.32 × 10-8 M). b Functional effects of the 92AA gD fragment on α7 nAChRs expressed in Xenopus oocytes. Oocytes were co-exposed to both ACh and the 92AA HSV1 gD fragment. AUC was determined for each trace and results of at least 4 oocytes were pooled to obtain the data shown in the figure. Representative traces show the inhibition of acetylcholine-induced responses on α7 nAChRs at three different concentrations (1.33 μM, 2.66 μM, 4 μM) of the 92 AA fragment of HSV1 gD produced AUCs of 3251, 2099, 1894 μA·ms, respectively. c Dose-response curve for inhibition of nicotinic induced responses by the 92 AA fragment HSV1gD. Responses obtained from at least 4 different oocytes were pooled to determine the inhibition dose-response curve for the 92 AA fragment inhibition of acetylcholine-induced responses on α7 nAChRs (IC50 = 2.68 µM, 95% CI: 2.19–3.27 µM).
The 92AA HSV1 gD ectodomain fragment was also evaluated for its ability to inhibit agonist-induced responses on α7 nAChR. Oocytes expressing α7 receptors were co-exposed to 100 µM acetylcholine (EC60) and the 92AA HSV1 gD ectodomain fragment at concentrations ranging from 0.44–8 µM (Fig. 3b, c). Similar to the whole ectodomain, this 92AA fragment inhibited α7 receptors when co-applied with acetylcholine, albeit with a lower IC50 (IC50 = 2.68 µM, 95% CI: 2.19–3.27 µM).
Binding and functional effects of HSV1 gD, 24AA putative binding loop peptide with nAChR
To determine if the proposed α-Bgtx homologous loop region was responsible for the binding of HSV1 gD to the L-AChBP and the functional effects observed on α7 nAChRs, a small, 24 AA peptide corresponding to the homologous loop region was used (Sequence: NAPSEAPQIVRGASEDVRKQPYNL). This peptide was evaluated using SPR and functional assays for the whole ectodomain and 92AA fragment of HSV1 gD.
In SPR, the 24 AA peptide from HSV1 gD was shown to bind to the L-AChBP with a Kd of 4.32 x 10-4 ± 3.99 x 10-4 M (Fig. 4a). This was a lower affinity than observed for the whole ectodomain or the 92 AA fragment.
a Binding to the L –AChBP, SPR binding data at five different concentrations (31 μM, 62 μM, 125 μM, and 250 μM) of the 24AA HSV1 gD peptide. L-AChBP is the ligand, and the 24AA HSV1 gD peptide is the analyte. The analyte was injected for 4 min at a flow rate of 25 μL/min. Binding data was fit to a 1:1 binding model (Kd = 4.32 × 10-4 ± 3.99 × 10-4 M.). b Functional effects of the 24 AA gD peptide on α7 nAChRs expressed in Xenopus oocytes. The 24AA fragment of HSV1 gD inhibited acetylcholine-induced responses of α7 nAChRs in a dose-dependent manner. Four different concentrations of the putative binding loop peptide (10 μM, 30 μM, 100 μM, and 300 μM) produced AUCs of 666, 634, 647, and 140 μA·ms respectively. c Dose response curve for inhibition of nicotinic-induced responses by the 24AA loop peptide of HSV1gD. Oocytes were co-exposed to 100 µM ACh and the 24AA peptide. AUC was determined for each trace, and results of at least 4 oocytes were pooled to obtain the data shown in the figure (IC50 = 157 µM, 95% CI:132 µM to 186 µM).
The 24AA peptide derived from the putative HSV1 gD binding loop (G83–P92) was also evaluated for its ability to inhibit acetylcholine-induced responses on oocytes expressing α7 nAChRs. Similar to the 92AA ectodomain fragment, oocytes expressing α7 receptors were co-exposed to 100 µM Acetylcholine (EC60) and concentrations of the 24AA peptide ranging from 10 to 300 µM. As with the 92AA fragment, the 24AA fragment inhibited the acetylcholine-induced response (Fig. 4b). The IC50 for the inhibition of 100 µM acetylcholine responses on α7 nAChRs was 157 µM (95% CI:132 µM to 186 µM) (Fig. 4c).
A summary of binding and functional data for the HSV1 gD whole ectodomain, 92 AA fragment protein, and 24 AA loop peptide is shown in Tables 1 and 2. Table 1 shows the binding affinity of each protein or peptide to the L-AChBP. The rank affinities of HSV proteins and peptides observed in these three experiments were: 92AA HSV1gD fragment >HSV1gD Whole ectodomain >24AA putative binding loop peptide. This rank affinity is equivalent to the relative on-rates as determined by k1 rate constant in these experiments, with the slowest binding occurring for the 24 AA putative binding loop peptide and the fastest for the 92AA fragment.
A comparison of the Ki values for inhibition of agonist responses by the HSV1 gD whole ectodomain, 92AA ectodomain fragment, and 24AA putative binding loop region on α7 nAChRs is shown in Table 2. The Ki was determined from the IC50 values using the Cheng Prusoff equation. The rank potency for these three molecules was whole ectodomain >92AA fragment >24 AA fragment. This was similar, but not identical to that obtained for SPR with the L-AChBP, but consistent with an expected decrease in potency for smaller, more flexible fragments.
Docking of HSV1 gD with α7/L-AChBP chimera
The binding and functional data described above indicate a likely binding site for HSV1 gD on the nicotinic receptor. In order to determine the location of potential binding sites, the web-based CADD modeling environment ezCADD was used36. The crystal structure of α7/L-AChBP chimera (PDB ID: 4HQP) was used as a model for the herpes1 gD docking study37. In the ezCADD software, the ezPPDock application was used for protein-protein docking. To validate the docking procedure, we re-docked the α-Bgtx molecule bound to the α7/L-AChBP chimera to determine if it could reproduce the crystallized structure38. The ezPPDock docking algorithm identified a potential interaction between the two subunits of an α7/L-AChBP chimera and HSV1 gD (Fig. 5). This algorithm produced a range of docking locations with the top 10 docking scores ranging from 3805.19 to 4597.61. Two thousand scores were analyzed using the Visual Molecular Dynamics (VMD) application, developed by the Theoretical and Computational Biophysics Group of UIUC39. Among these, pose 4, with a numerical score of 3970.30, shows the homologous loop of HSV1 gD (G83–P92) docked near binding Loop C of the α7/L-AChBP chimera. Pose 4 closely matches the binding of α-Bgtx (C29-V40) to the α7/L-AChBP chimera.
a Docking of HSV-1 gD (red) with α7/L-AChBP chimera (blue). Pose 4 indicates an interaction at the interface between subunits, near the orthosteric binding site. b Detail, showing α7/L-AChBP subunits chain A (blue) and chain B (orange) docked with HSV1 gD (red). The region labeled α1’ indicates the α-BgTx homologous binding loop of HSV1 gD (G83–P92).
Using pose 4 from the ezCADD docking study, a VMD contact map analysis indicates that α7/L-AChBP chimera chain A amino acids Y184, E185, and C186 are in close contact with R89, K90, and Q91 of the HSV1 gD homologous loop region (Fig. 6a). In addition, α7/L-AChBP chimera chain B amino acids G157, E158 are in close contact with amino acids V81, G83, A84, S85, V88, R89 located near the HSV1 gD homologous loop region (Fig. 6b).
Ligand and receptor amino acids within the binding region are placed on the X-axis and Y-axis. All amino acid residues that are in close contact will appear on the map with dark shading. More distant contacts are visualized as lighter shades. a VMD contact map between α7/L-AChBP chain A and HSV1 gD ectodomain (pose 4). The contact map indicates close contact between residues in α7 chimera chain A (Y184, E185, and C186) and the HSV1 gD loop region (R89, K90, Q91). b α7/L-AChBP chimera chain B alignment with HSV1 gD ectodomain. The contact map shows interacting residues between α7 chimera chain B amino acids (M156, Q157, and E158) are in close contact with the HSV1 gD amino acids I80, V81, G83, A84, S85, V88, R89.
HSV1 gD interaction with α7/L-AChBP chimera: Maestro and VMD analysis
The PDB file for pose 4 of HSV1 gD docked into the α7 nAChR/AChBP chimera was uploaded into the Maestro workspace. Further evaluation of protein-protein interactions identified the crucial amino acids involved in the interactions between HSV1 gD and the α7/L-AChBP chimera. HSV1 gD loop (G83-P92) (red) and α7 nAChR chimera chain A (blue) are depicted in (Fig. 7). HSV1 gD amino acid K90 is in close proximity to Y184 and C186 of α7/L-AChBP chain A, Loop C region (S180 -P190), likely interacting via a cation-pi interaction. HSV 1gD R89 and P92 closely interact with Q185 of α7/L-AChBP chain A. Also, HSV1 gD K90 could be involved in a cation-pi interaction with Y191 of α7/L-AChBP chain A.
Left Key interacting residues between HSV1 gD loop region (G83 - P92) (red) and α7/L-AChBP chimera chain A Loop C (blue). Right Interacting residues between HSV1 gD and chain A of α7/ L-AChBP chimera viewed from a 180° rotation around the vertical axis of the left figure. HSV1gD amino acid K90 is involved in a cation-Pi interaction with Y184 and C186 amino acids of nAChRs chain A Loop C region.
On the other hand, HSV1 gD amino acid S85 closely interacts with amino acids Q55 and Q157 of α7/L-AChBP chain B likely via hydrogen-bond. In addition, HSV1 gD amino acids D87 and E210 are in close proximity to Q114, L116, Q133 amino acids of α7/L-AChBP chain B.
Discussion
The specific hypothesis of this project was that viral glycoproteins possess a common structural loop region that enables them to bind nicotinic acetylcholine receptors and that this loop region is structurally homologous to the binding loop of Ly6 endogenous proteins as well as the 3-fingered neurotoxins typified by α-Bgtx. We demonstrated that HSV1 gD, along with other similar viral proteins, contains a region possessing significant structural homology to the binding loop of LY6 proteins and the primary binding loop of α-Bgtx. The ectodomain of HSV1 gD can inhibit nAChRs, and smaller fragments of this ectodomain containing the homologous binding region also appear to bind to nAChRs, albeit with lower affinity. While the HSV1 gD and fragments inhibited agonist induced responses, at very low concentrations, some degree of potentiation was observed on the α7 receptor. This phenomenon was observed with α7 receptors at the lowest HSV1 gD concentration (Fig. 2b). This type of potentiation in the presence of low concentrations of antagonist is common in fast desensitizing nAChRs such as the α7 receptor40. α7 receptors undergo a fast transition between different states including resting, active, and desensitized conformations during signaling. Each of these states shows different affinities for different nAChR ligands41. One explanation for the potentiation observed at low concentrations of antagonists is that they may alter the desensitization kinetics of the receptor, thereby increasing the apparent agonist response42.
We have further explored the mechanism of this interaction and the similarity of this binding region to α-Bgtx binding using computational docking studies that demonstrate a high likelihood that α-Bgtx and HSV1 gD share a common binding domain. Docking studies using structures of the HSV1 gD and an α7/L-AChBP chimera identified a putative binding domain for HSV1 gD that overlaps the orthosteric binding site for agonists. Thus, homology studies, SPR, functional studies, and docking, all support an interaction between nAChRs and HSV1 gD. Since the docking study showed that HSV1 gD and α-Bgtx share the same orthosteric binding site on α7 nAChR, this suggests the possible competitive inhibition of the receptor functions by HSV1 gD. This was the basis for using the Cheng-Prusoff equation to calculate inhibition constants. The lack of sufficient HSV1 gD prevented us to perform a competitive binding assay using the known orthosteric ligands for α7 nAChR. In the future, functional studies can be done to confirm the competitive inhibition.
The structural homology alignment predicted that K90 and Q91 may be important amino acids for the interaction of HSV1 gD. Docking and VMD contact map analysis indicates that Y184, Q185, and C186 of the α7/L- AChBP chimera are in close proximity to K90 and Q91 of HSV1 gD, forming a likely cation-pi interaction between K90 and Y184. Y184 is a key residue in ligands binding to the nAChR. The cation-pi interaction’s strength, at this point, is likely a significant contributor to the binding of the viral protein. Similar interactions are common for nicotinic ligands. A similar interaction is possible in the L-AChBP, thus explaining the ability of HSV1 gD to bind to the L-AChBP, confirmed by SPR experiments.
A critical amino acid in α-Bgtx, R36, is known to form a cation-pi interaction with Y184 of the α7 nAChR. From our structural homology data, K90 of HSV1 gD may also form a cation-pi interaction with Y184 and is likely to be a key interaction at this position. Further confirmation of this exists in the SARS-CoV-2 spike protein, S1, which contains R82 at this position, and the Rabies virus glycoprotein, which includes a R97 (based on sequence alignments with α-Bgtx)26. Therefore, K90 is likely to be a critical amino acid in the binding of HSV1 gD to the α7 nAChR. This significant finding could lead to identifying other viruses that have potential interactions with nAChRs.
The structural similarity between glycoproteins from several virus families suggests that the nAChR binding motif may be a common feature of some viruses. Along with recent investigations into SARS-CoV2 interaction with nAChRs and prior research implicating interactions of gp120 of the HIV and the neurological effects of HIV, our data supports this hypothesis43. The evolution of viruses to modulate nAChRs would enable them to potentially regulate inflammation and immune system function, thus helping them evade an immune response and increase viral replication. With respect to HSV1, studies have shown that activation of α7 nAChRs can impact the neuropathology of HSV44,45. More recent work has shown that activation of α7 nAChRs appears to inhibit HSV1 replication in microglia via modulation of IL-1B and Nos2 among others46. Inhibition of the α7 nAChR, as shown in our study, would be expected to produce the opposite effect of activation, thus enhancing viral replication and pathogenesis. If this ability to inhibit nAChRs is a common feature of other viruses, the role of α7 nAChR in viral pathogenesis could provide a novel approach to treatment.
As a result of these nicotinic receptor actions in the immune system, viruses capable of entering the CNS could produce neurological effects by interacting with similar neuronal receptor subtypes. In addition to identifying viruses with potential neurological effects, there may be some opportunities for therapeutic intervention by targeting these receptors with recently identified pharmacological agents. Nicotinic receptor positive allosteric modulators (PAMs) could potentially be used to combat virus-induced neurological symptoms. PAMs enhance nAChR activity in the presence of a receptor agonist. It has been shown that stimulation of nAChR activity via a selective PAM resulted in the displacement of Aβ protein (implicated in AD progression) from nAChRs47. Enhancing nAChR activity could also potentially increase neuronal signaling, compensating for viral inhibition and stimulating the anti-inflammatory effects of nAChRs. Other potential drugs that may combat these effects include the smoking cessation drugs nicotine and varenicline. Further study is needed to elucidate a common mechanism of how viruses such as HSV1 may impact the cholinergic system and how these effects may contribute to their known neurological effects.
In conclusion, HSV1 gD contains a structural loop with apparent homology to LY6 proteins and the three-finger toxins (3FTx). Arginine (R36) in the 3FTx binding domain was identified as a crucial amino acid for the α-Bgtx. interaction with the Loop C of α7 nAChRs48. Using α-Bgtx as a model, a computational modeling study suggested that Lysine (K90) of HSV1 gD is crucial to interact with the Loop C of nAChRs. This data provides information that can be used to potentially identify other viruses that may interact with nAChR and contributes to ongoing studies implicating viral interaction with nAChRs as a common pathway for neurological effects of viruses.
Among the viruses that may use a similar nAChR binding motif are Rabies, HIV, and more recently SARS-CoV2. Based on structural and sequence similarity between the RVG and α- Bungarotoxin (α-Bgtx), the rabies virus is thought to bind to the acetylcholine binding site of the muscle nAChRs49. Additionally, the COVID-19-causing virus SARS-CoV-2 also appears to contain a neurotoxin-like region proposed to enable the virus to act on nAChRs, including the highly expressed α7 and α4β2 subtypes50,51. However, there have been contradictory reports regarding SARS-CoV-2 spike protein interactions with nAChRs. Computational modeling data predicted that SARS-CoV-2 spike protein S1 contained a neurotoxin-like region (Y674-R685) that enables the virus to have potential interactions with nAChRs52. A more recent study has refuted these claims. The SARS-CoV-2 spike protein S1 interactions with α7 nAChRs were investigated using ligand binding and electrophysiology assays, but the data did not suggest spike protein interactions with nAChRs53. Unfortunately, the SARS-CoV-2 Spike protein S1 neurotoxin-like region (Y674-R685) is not visible in crystal structures, likely due to its flexibility in the crystal. It is possible that the Spike protein S1 neurotoxin-like region may not share a structural motif with 3FTx even though it contains some sequence similarities. Our data could be effectively used in modeling the missing structure of the SARS-CoV-2 spike protein if its nAChR interaction could be confirmed. Mutation of the spike protein could provide this information.
In addition to identifying other nAChR-binding viruses, a possibly more important outcome of this research is the identification of a potential mechanism by which neurotropic viruses may be related to Alzheimer’s disease. Alteration in the expression of different nAChR subtypes is critical to AD development, particularly involving α7 receptors54. Prior research has suggested a strong link between viruses such as HSV1 and AD progression55,56,57. However, specific mechanisms have not been forthcoming, aside from the idea that general inflammation may be connected to the development of AD. Other disorders, such as Autism, have also been shown to increase after exposure to viruses such as influenza58. Likely, long Covid and Chronic Fatigue Syndrome (Myalgic encephalomyelitis) are also a result of unknown neurotropic viral mechanisms. Our findings, for the first time, suggest a mechanism by which HSV1 could produce its neurotropic effects: the specific targeting and inhibition of nAChRs. The resulting disruptions in cholinergic function may help explain the subsequent neurodegenerative effects of this virus, including memory effects and cognitive dysfunction. The known periodic re-activation of HSV1, often during stress, would likely exacerbate this condition, possibly producing cumulative effects over time.
As discussed above, a large number of neurodegenerative diseases involve dysfunction of the cholinergic transmission system in the CNS and PNS, and hence nAChRs are potential therapeutic targets for the treatment of multiple neuropathological diseases59,60,61. Our data, along with those implicating other viruses such as SARS-CoV-2, suggests a novel mechanism of action for neurotropic viruses. More viruses need to be examined to define the structural motif that enables a virus to inhibit nicotinic receptors. The research presented here lays the groundwork for those studies. Virus interactions with nAChRs could be a common mechanism of viruses for their pathogenesis in the nervous system and potential immune system effects.
Materials and methods
Structural homology
DNASTAR Navigator-17 Lasergene software®, supported by the ISU Pharmaceutical Sciences Computational Core Laboratory, was used for structural and sequence homology studies62. DNASTAR’s integral Protean 3D application was used for structural homology searches and analysis. The protean 3D tool is used to predict protein’s secondary structures, identify functional domains, and perform a structure modeling32. Two alignment methods, TM-Alignment and jFATCAT were used for structural analysis to compare a selected region of HSV1 glycoprotein D with α-bungarotoxin. TM-Align measures the structural similarity between two protein structures by considering the similarity of their alpha carbons63. It calculates residue-to-residue alignment based on protein structure similarity. Quality of fit is determined using the RMSD (Root mean square deviation) between two protein structures64. RMSD measures the distance of all the residues, but it also depends on the length of the protein65. DNASTAR offers a TM-Align rigid algorithm that measures the distance of the atoms between the superimposed protein structures. It does not consider the flexibility of the backbone63.
FATCAT (Flexible structure Alignment by Chaining Aligned fragment pairs allowing Twists) is used for protein structure alignment. This allows flexible alignment considering local and global structural similarities66. DNASTAR offers a jFATCAT (Java-based Flexible structure Alignment by Chaining Aligned fragment pairs allowing Twists) rigid algorithm. jFATCAT rigid alignment is similar to TM-align, which performs alignment by superimposing the backbone of the two protein structures. jFATCAT does not allow conformation flexibility, but it offers additional features like improved visualization67.
Protein database (PDB) IDs of α-bungarotoxin (1HC9) and herpes1 gD (2C36) structures were imported into the Protean 3D application workspace68. We performed protein structural homology modeling of a selected loop region from the herpes virus glycoprotein and α-bungarotoxin 3FTx loop region (C29 - V40) to perform the alignment procedure. Both TM-align and jFATCAT methodologies were used to perform structure alignment between the selected loops from both proteins.
The MegAlign Pro 17 application performed sequence alignment between desired protein sequences69. Protein FASTA sequence files from the PDB IDs of α-bungarotoxin (1HC9) and the HSV1 gD (2C36) were imported into the MegAlign Pro 17 application for protein sequence alignments. We performed sequence homology using four different methodologies: Clustal Omega, Clustal W, MAFFT, and MUSCLE, which are available in the MegAlign Pro 17 application.
SPR methodology
The Limnea-acetylcholine binding protein (L-AChBP) was used as the ligand in SPR studies and had been previously synthesized in Dr. Schulte’s laboratory at the University of Alaska Fairbanks. Verification of L-AChBP was determined by LC-MS method for its physical stability70. A PAGE protein gel run confirmed the presence of assembled AChBP71.
Tubocurarine chloride, an nAChR antagonist, was used as a control drug to validate the quality of the immobilized L-AChBP chip surface. Tubocurarine hydrochloride pentahydrate (Sigma Aldrich®, cat #T2379) is an antagonist to the nAChRs. Tubocurarine was used as a control drug to characterize the AChBP SPR chip. Tubocurarine binding was identical to that previously determined by the Schulte laboratory. Once an appropriate quality SPR surface was prepared, SPR analysis of viral protein binding to the AChBP was determined.
For HSV1 gD, binding was determined for (1) the entire ectodomain, (2) a fragment containing the putative binding loop (92 AA), and (3) a small peptide of only the putative binding loop (24 AA). The HSV1 gD whole ectodomain was commercially synthesized by My BioSource® (Cat # MBS319239). A fragment of the HSV1 gD ectodomain consisting of 92 amino acids was also commercially obtained from My BioSource® (Cat # MBS147142). The 24AA peptide, corresponding to the putative HSV1 gD binding loop, was synthesized in the Pharmaceutical core laboratory in the Biomedical and Pharmaceutical Sciences department at ISU and characterized by HPLC (purity >90%).
The 24AA HSV1 gD peptide was synthesized through a standard method of Fmoc-mediated solid-phase peptide synthesis (SPPS) using Aapptec Focus Xi peptide synthesizer (Louisville, KY, USA). The coupling reagents, solvents, Fmoc-protected amino acids, Wang and 2- chlorotritylchloride (2-CT) resins were purchased from Aapptec (Louisville, KY, USA). A 5-fold excess of amino acids and coupling reagents were required compared to the first amino acid. The chain elongation was performed by deprotecting the Fmoc group from the resin-attached amino acid by 20% Piperidine in DMF. The next Fmoc-protected amino acid was activated by diisopylcabodiimide (DIC) and Oxyma as a nucleophilic addition, followed by coupling for an hour. The deprotection, activation and coupling process were repeated until the entire peptide was synthesized. After completion of synthesis, the last Fmoc group was de-protected and cleaved from the resin by 92.5% of Trifluoroacetic acid (TFA), 2.5% of Triisopropyl silane (TIS), 2.5% 2,2-Ethylamine ethanediol and 2.5% of water. Peptide purity was checked using an Agilent Technologies 1220 Infinity II LC DAD system in a linear gradient from 0% to 60% of 0.1% TFA in acetonitrile on an Agilent Eclipse C18 column (4.5 X 150 mm,5 µm) for 10 min. HPLC-grade solvents and reagents were purchased from Sigma Aldrich (St. Louis, MO).
The synthesized peptide was characterized by electrospray ionization technique in an AB SCIEXQTRAP® 5500 mass spectrometer (Foster City, CA, USA) in tandem with Nexera HPLC system from Shimadzu Corporation (Columbia, MD). Analyst software was used for data acquisition and data processing. PEPCALC and Skyline software were used as references to determine the total mass of the peptide and the transition of peptides, respectively.
The tubocurarine binding study confirmed that the L-AChBP was active while immobilized on the chip. HSV1 gD whole ectodomain and 92 AA protein fragments were commercially synthesized from My BioSource.
L- AChBP was immobilized onto the CM5 sensor chip using a standard amide linking procedure. The flow rate was consistently maintained at 25 μl/min during immobilization. L-AChBP was immobilized, and the unreacted chip surface was blocked by reaction with ethanolamine. The immobilized AChBP density was typically about 7000 resonance units (RU). For L- AChBP immobilization, pH scouting was conducted without modifying the sensor chip surface (CM5 chip surface). Lyophilized L- AChBP was re-hydrated in 200 μL of PBST to create a 5 mg/mL stock solution. Subsequently, 300 μg/mL L- AChBP solutions were prepared in 10 mM sodium acetate buffer ranging from pH 3.0–5.5. Using a PBST running buffer (37 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 4.5), a non-activated chip was exposed to these solutions to determine the optimal pH for interaction of L- AChBP with the surface.
Following the pH scouting study, the CM5 chip was activated by application of 200 μL of 0.4 M EDC and 0.1 M NHS for 8 min across both flow cells. L- AChBP at a concentration of 300 μg/mL was then introduced into flow cell 1 for an additional 8 min to immobilize AChBP on the chip surface. Both flow cells 1 and 2 were then exposed to 1 M ethanolamine at pH 8.5 for 8 min to block all activated carboxymethyl groups on the sensor surface. This created an active AChBP chip surface in flow cell 1 and a blocked, non-AChBP surface in flow cell 2, permitting subtraction of non-specific binding during binding experiments. A typical AChBP density was achieved of about 7000 resonance units (RU), sufficient for conducting glycoprotein binding studies.
Herpes1 glycoprotein D (HSV1 gD) whole ectodomain and peptide fragments used as the analyte were dissolved in 10 mM PBST buffer (37 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 4.5). Glycoproteins were diluted with 10 mM PBST to a series of different concentrations then introduced into the chamber at a flow rate of 25 μL/min. The change in the refractive index of the metal film due to the interaction of the ligand & analyte produced a change in the SPR signal, generating a sensogram. The SPR response is proportional to the number of analyte molecules bound to the L-AChBP chip surface.
Method for functional study
The DNA sequence for the human α7 nAChR (NM_000746.4) subunit was inserted into the pcDNA3.1-CHRNA7/amp vector using the XhoI and EcoRI restriction sites located in the polylinker. The DNA sequence for the molecular chaperone Ric-3 was similarly inserted into pcDNA3.1 + /C-(K)DYK/amp using the BamHI restriction site. Ric-3 (NM_001206671.4) has been shown to increase expression of α7 nAChRs in Xenopus oocytes72. Plasmid DNA was produced using e. coli and purified DNA purified using Qiagen plasmid purification kits. The purified cDNA was characterized using gel electrophoresis. The cDNA for α7 and ric3 were characterized using digests with the restriction enyzmes SspI and FspI. Capped cRNA transcripts for each nAChRs subunit were prepared by in-vitro synthesis using the Invitrogen T7 mMESSAGE mMACHINETM transcription kit (cat # AM1344).
Acetylcholine and nicotine were purchased from Tocris Bioscience for the functional study conducted in Xenopus oocytes. HSV1 gD whole ectodomain and a fragment of HSV1 gD ectodomain of 92AA were commercially synthesized by My BioSource (Cat # MBS147142). A small peptide of 24 amino acids from the HSV1 gD loop was also synthesized by My BioSource® to a purity of >95% (determined by HPLC analysis).
The two electrode voltage clamp (TEVC) electrophysiology technique is an established approach for the study of ion channel receptors73. This approach provides information on the functional of receptors and their modulation by endogenous and exogenous agents. TEVC enables the identification of these agents as agonists, antagonists, or allosteric modulators and allows the use of expressed human receptor subtypes74.
Xenopus oocyte preparation and protein expression
This study uses Xenopus oocytes to express α7 nAChRs, and TEVC electrophysiology to determine the functional effects of HSV1 gD on α7 nAChRs. Xenopus laevis oocytes state IV and V were purchased from Xenopus1 Inc. Oocytes were digested with 2 mg/ml collagenase I in Barth’s buffer [88 mM NaCl, 1 mM KCl, 0.4 mM CaCl2, 1 mM MgCl, HEPES 10 mM, pH 7.4] containing 50 mg/L gentamycin. Oocytes were shaken gently in this solution for 1–1.5 h at room temperature. Digested oocytes were then washed, sorted, and stored overnight in Barth’s buffer [88 mM NaCl, 1 mM KCl, 0.4 mM CaCl2, 1 mM MgCl, HEPES 10 mM, pH 7.4] with 1 mM pyruvate at 16 oC. After resting overnight, about 50–60 oocytes were injected with 46 nl of a mixture of α7 and ric3 cRNA to express respective receptors in oocytes. Prior to injection, α7 and ric3 cRNA were mixed at a concentration of 1200 ng/ul and 400 ng/ul respectively. Functional α7 were expressed and utilized within 5–10 days of injection. Prior to recording, the follicle layer on the oocyte was removed using fine forceps.
Two Electrode Voltage Clamp (TEVC)
TEVC recording was performed using an automated oocyte recording system utilizing a Gilson 231 autosampler/injector for sample delivery. The system utilizes an OC-725C oocyte clamp amplifier (Warner Instruments, CT, US) coupled with a computerized data acquisition system (Digidata 1550B, Molecular Devices LLC). Data was collected using Clampex Software and analyzed using Clampfit software (Molecular Devices LLC). Oocytes were held in a 100ul volume chamber and clamped at holding potential of -60 mV and perfused with an ND96 recording buffer (96 mM NaCl, 2 mM KCl,1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES pH 7.4) throughout the experiment. Recording and current electrodes were pulled from thin wall glass capillary tubes (Warner instruments, cat. # 740771) and filled with 3 M KCl. Electrodes with resistance of 1–5 MΩ were chosen for use in these studies. All drugs and 56 peptide solutions were prepared with ND96 recording buffer.
For herpes protein (HSV1 gD whole ectodomain and small peptide fragments) studies, Xenopus oocytes injected with the α7 and ric3 cRNA combinations were exposed to agonist acetylcholine/nicotine. An agonist dose response was determined to confirm the expression of the α7 nAChRs. To determine the functional effect of herpes proteins on agonist induced currents, oocytes were pre-exposed to each protein for 30 s prior to co-application with an agonist (acetylcholine or nicotine) for another 30 s. Peak responses from at least 4 oocytes obtained from different frogs were combined and plotted against the concentration of either viral protein concentration (viral inhibition curve) or agonist concentration (dose/response curve). For α7 receptors, rapid desensitization and re-activation often produced multiple peaks during the response. Since this prevented the use of a single peak response amplitude to quantify the response, a total current calculation was used for these receptors. The total current flowing through the channel during opening is reflected in the area under the response curve (AUC). The AUC was calculated using Clampfit software (Molecular Devices®) and this value was used to calculate the total response during agonist or agonist/antagonist application on α7 receptors. Dose/response data were plotted and analyzed using graphpad prism software®. Nicotine was typically used as the agonist when evaluating viral protein effects on α7 receptors since nicotine produced less desensitization and fewer multiple peaks, thus providing a better quantitative analysis of the effects.
To determine if viral proteins could produce a response in the absence of an agonist, proteins were applied alone to nAChR-expressing oocytes. None of the proteins tested could elicit responses when applied alone on α7 receptor.
Data analysis
Graph Pad® Prism, version 10 was used to analyze the functional data obtained in TEVC studies. All responses from a single oocyte were normalized by the response to an oocyte at a fixed concentration of an agonist. This provided a means to compare responses between heterologously expressed receptors from different oocytes. Nicotine-induced responses of α7 nAChRs were normalized to the response of an oocyte to a concentration equal to the EC40 for the agonist nicotine. Using nicotine EC40 allowed us to perform experiments with lesser amounts of herpes protein, i.e. less amount of herpes protein was needed to inhibit nicotine-induced response. For experiments using acetylcholine stimulation of α7 nAChRs, responses were normalized to the response of an oocyte to concentrations equal to the EC60 for acetylcholine. To evaluate the stability of the oocyte response over time, oocytes were exposed to this concentration of agonist periodically throughout the recording period. To ensure the reproducibility and precision of the data, experiments were repeated a minimum of 4 times with oocytes from at least 4 frogs.
Inhibition curves were produced by plotting the percent of the normalized response remaining when co-exposed to different concentrations of protein or peptide. Using standard inhibition algorithms in Graphpad Prism, a non-linear regression curve fitting method was used to fit inhibition data for α7 receptors. This enabled the determination of the IC50 along with appropriate confidence intervals. A 100 percent response to agonist was determined by pooling data for the application of the test concentration of agonist to multiple oocytes. The application of glycoproteins produces either potentiation (increase from 100%) or inhibition (decrease from 100%). The percent change in the response due to different concentrations of peptides (Y-axis) was plotted as the “percent response” vs. the concentration of the test protein or peptide (Y-axis). For most experiments, response data was constrained during curve fitting to 100% for the highest percent response (no inhibition) and 0% for the lowest percent response (100% inhibition). At low peptide concentrations, all HSV1 gD proteins and peptides showed an initial increase (slightly >100% response) followed by inhibition of the agonist response. A 95% confidence interval was calculated for the determined IC50 and Hill slope. An R2 value was determined to quantify how well the data fit the regression model used.
The Cheng Prussoff equation was used to calculate an approximate Ki for inhibition data to compare data obtained for different inhibitors.
Docking analysis
Protein-protein docking was performed using Maestro (Schrodinger®) and ezCADD (Xu et al.) software75,76.
For docking of HSV1 gD, both the HSV1 gD protein and the α7/L-AChBP were prepared using Maestro® software by Schrödinger. The preparation of ligand (herpes1 glycoprotein D) and receptor (α7 nAChR/L-AChBP chimera) for docking also used Maestro. The ezPPDock application was used to perform protein-protein docking. The ligand and receptor pair were imported into the workspace of Maestro 13. The protein preparation workflow included the pre-processing and optimizing protein structures for protein-protein docking. This process involves removing water molecules, filling up missing residues, and optimizing hydrogen bonds.
In the ezCADD software, the ezPPDock application was used for protein-protein docking. The α7/L-AChBP chimera structure (PDB ID: 4HQP) was imported into the ezPPDock workspace. Chimeric receptor chains C, D, and E were blocked in the region of (Q3-G203) to restrict the α-Bgtx molecule from binding to the inside of the receptor channel domain, directing it towards the outside surfaces of the receptor. Without putting any restrictions on the binding domain structure, the α-Bgtx molecule was allowed to dock into the α7/L-AChBP chimera chains A and B. The resolved crystal structure showed the α-Bgtx (chain I) molecule bound to α7/L-AChBP chimera chains A and B. To validate the docking method, α-Bgtx (chain H) was docked with the α7/L-AChBP chimera chain A and B using the ezPPDock application. The α-Bgtx (chain H) is the furthest chain from the α-Bgtx chain I. After successfully re-docking the α-Bgtx molecule into the α7/L-AChBP chimera, we moved forward with the HSV1 gD docking into the α7/L-AChBP chimera.
Docking of HSV1 gD to the α7/L-AChBP chimera was performed using ezPPDock. No restriction or blocking of binding domains was applied to HSV1 gD, although binding to the α7/L-AChBP chimera was restricted to bind to chains A and B only.
HSV1 gD (PDB ID 2C36) and α7/L-AChBP chimera (PDB ID 4HQP) docking results were analyzed using the VMD application. PDB files for the α7/L-AChBP chimera (receptor) and HSV1 gD (ligand) were uploaded to the VMD workspace. The DCD file containing the docking results obtained using the ezPPDock application was also uploaded to the VMD workspace. The resulting analysis indicated many potential ligand-binding poses obtained from the docking studies. This docking method also produces a scoring method to rank the best docking positions.
The best pose was chosen by comparing the predicted binding of the HSV1 gD region (G83-P92) with the binding of α-Bgtx 3Ftx (C29–V40) in the crystal structure. Pose number 4, of the top 10 ranked poses, was identified as the best pose within this region since it bound to a similar location to α-Bgtx in the orthosteric binding. A contact map of potential interactions was calculated for this pose. The contact map enables visualization of the potential ligand/receptor interactions.
Data availability
AChBP cDNA was synthesized using a published sequence (https://www.uniprot.org/uniprotkb/P58154/entry). HSV-1 gD whole ectodomain and fragments were synthesized using the published sequence (https://www.uniprot.org/uniprotkb/Q69091/entry). Human α7 nAChR DNA sequence is available at NCBI (https://www.ncbi.nlm.nih.gov/nuccore/NM_000746.4/). The Ric-3 cDNA sequence is available at NCBI (NM_001206671.4) (https://www.ncbi.nlm.nih.gov/nuccore/1519311567). α-bungarotoxin protein structure and sequence are available in the Protein database (PDB ID: 1HC9). Herpes1 gD protein structure is used from the Protein database (PDB ID: 2C36).
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Acknowledgements
This work was supported by an Idaho INBRE-4 Graduate Research Assistantship Grant and a grant by the Infectious Diseases Society of America (IDSA). This research also acknowledges the support from the ISU College of Pharmacy, Department of Biomedical and Pharmaceutical Sciences (BPSCI).
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M.S. conceived the study. S.Y. prepared the manuscript. K.S. performed binding study experiments. S.Y. did the study design and performed in-silico and in-vitro experiments. C.N. and D.X. designed and performed docking study. A.H. and A.R. helped with 24AA peptide synthesis. L.D. helped with the oocyte functional study. Y.A. synthesized the L-AChBP protein. M.S. and S.Y. performed data analysis and manuscript editing.
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Yeasmin, S., Sharma, K., Nicolet, C. et al. HSV1 glycoprotein D utilizes an LY6-like binding domain to inhibit alpha7 nicotinic receptors. npj Viruses 3, 52 (2025). https://doi.org/10.1038/s44298-025-00109-w
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DOI: https://doi.org/10.1038/s44298-025-00109-w
