
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39223222
71477
10.1038/s41598-024-71477-1
Article
Computational investigation in inhibitory effects of amantadine on classical swine fever virus p7 ion channel activity
Chen Xiaowei 12
Wang Xiao wangxiao2037@126.com

1
1 https://ror.org/008w1vb37 grid.440653.0 0000 0000 9588 091X School of Basic Medical Sciences, Binzhou Medical University, Yantai, 264003 China
2 https://ror.org/008w1vb37 grid.440653.0 0000 0000 9588 091X Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, 264003 China
2 9 2024
2 9 2024
2024
14 2038721 2 2024
28 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Classical swine fever virus (CSFV) p7 viroporin plays crucial roles in cellular ion balance and permeabilization. The antiviral drug amantadine effectively inhibits viral replication by blocking the activity of CSFV p7 viroporin. However, little information is available for the binding mode of amantadine with CSFV p7 viroporin, due to the lack of a known polymer structure for CSFV p7. In this study, we employed AlphaFold2 to predict CSFV p7 structures. Subsequently, we conducted a docking study to investigate the binding sites of amantadine to CSFV p7. Computational analysis showed that CSFV p7 forms a pore channel in a hexameric structure. Furthermore, molecular dynamics (MD) simulations and mutant analyses further suggest that CSFV p7 likely exists as a hexamer. Docking studies and MD simulations showed that amantadine interacts with the hydrophibic regions of tetramer and pentamer, as well as with the hydrophobic pore channel of the hexamer. Considering the potential hexameric assembly of CSFV p7, along with docking results, MD simulations, and the characteristics of the gated ion channels, we propose a model of CSFV p7 ion channel based on its hexameric configuration. In this model, residues E21, Y25, and R34 are suggested to selectively recruit and dehydrate ions, while residues L28 and L31 likely act as hydrophobic constrictors, thereby restricting the free movement of water. The binding of amantadine to residues I20, E21, V24 and Y25 effectively blocks ion transport. However, this proposed molecular model requires experimental validation. Our findings give a structural insight into the models of CSFV p7 as an ion channel and provide a molecular explanation for the inhibition effects of amantadine on CSFV p7-mediated ion channel conductance.

Keywords

Classical swine fever virus
p7 viroporin
Ion channel
Amantadine
Binding sites
Subject terms

Target identification
Antivirals
Virus structures
Protein structure predictions
Binzhou Medical Universityissue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Classical swine fever virus (CSFV) is the etiological agent responsible for causing a highly contagious and economically significant viral disease in both domestic and wild pigs. Alongside bovine viral diarrhea virus (BVDV) and border disease virus (BDV), CSFV is classified as a member of the genus Pestivirus within the family Flaviviridae1,2. The genome of CSFV spans approximately 12.3 kb in length and comprises a single open reading frame that encodes a polyprotein consisting of 3,898 amino acids. Following the polyprotein cleavage, the resultant process generates 12 individual proteins in the following order: Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B3–5.

Viroporins, such as the influenza A virus (IAV) M2, hepatitis C virus (HCV) p7, human immunodeficiency virus (HIV) Vpu, and picornavirus 2B, are known to form channels and associate with host membranes6,7. These viroporins play a critical role in modulating the cellular ion balance and facilitating permeabilization to facilitate viral entry, assembly and release. CSFV p7 protein, also as a viroporin, is highly involved in CSFV virulence and is indispensable for efficient virus production in host cells8,9. In additionally, CSFV p7 regulates viral replication by interacting with host cellular proteins, such as microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), and calcium-modulating cyclophilin ligand (CAML, also called CAMLG)10,11. Structurally, the CSFV p7 structure is still unknown though two transmembrane helices linked by a hydrophilic segment were predicted8. In contrast, the HCV p7 protein forms an ion channel as a hexamer and contributes to virion assembly12. Given the similarities within the Flaviviridae family, CSFV p7 protein is also likely to assemble in a hexameric arrangement to exhibit ion channel activity.

Various channel blockers or inhibitors can effectively block the channel activity of viroporins, which represents ideal targets for therapeutic intervention13. Several compounds were reported to interfere with the ion channel activity of viroporins and thereby inhibit virus production. Antiviral drugs such as amantadine and rimantadine specifically target the tetrameric proton channel of the IAV M2 protein, effectively inhibiting IAV replication14,15. Similarly, amantadine and long-alkyl-chain iminosugar derivatives have been shown to block the ion channel of the HCV p7 protein16,17. The drug-binding sites for IAV M2-amantadine and HCV p7-amantadine have been well characterized using nuclear magnetic resonance (NMR) technology12,14. Amantadine has also been demonstrated to inhibit CSFV replication by targeting the channel activity of CSFV p79,18. Furthermore, amantadine displayed a concentration-dependent inhibitory effect on CSFV replication, with effective inhibition observed at concentrations ranging from 125 to 500 μM8. Notably, amantadine resulted in a 100-fold reduction in viral yield at a concentration of 500 μM. Subsequent studies further indicated that amantadine effectively inhibited CSFV replication within a concentration range of 0 to 5 mM9. However, the 3D structure of CSFV p7 is unknown, which challenges the understanding of the mechanism by which amantadine blocks this process.

The recently developed AlphaFold2 algorithm is a powerful tool that accurately predicts protein structures. It has demonstrated high accuracy competitive with experimental structures in the majority of cases. The AlphaFold Protein Structure Database has been well established and almost covers the complete human proteome. In this study, we employed AlphaFold2 to predict the structures of CSFV p7 for the investigation of the binding sites of amantadine to CSFV p7. Our results showed that the structures of CSFV p7 were well predicted by AlphaFold2, revealing the assemble of the pore-forming channel in hexameric models. Subsequently, the structural models were docked with the amantadine by AutoDock Vina, resulting in the identification of drug binding regions. Combining the potential hexameric assembly of CSFV p7, docking results, MD simulations, and the characteristics of the gated ion channel, a model of CSFV p7 ion channel was proposed based on its hexameric configuration. In this model, residues E21, Y25, and R34 are suggested to selectively recruit and dehydrate ions, whereas residues L28 and L31 act as hydrophobic constrictors, restricting the free movement of water. The binding of amantadine to residues I20, E21, V24 and Y25 effectively blocks ion transport. However, this model requires experimental validation. In summary, our findings provide a structural model and molecular explanation for the ion channel conductance mediated by CSFV p7, as well as the inhibitory by amantadine.

Results

Conservation and hydrophobicity of CSFV p7 protein

The amino acid sequences of CSFV p7 from ten different CSFV isolates were analyzed (Fig. 1A), indicating a high degree of conservation among different CSFV strains. In addition, the amphipath of the CSFV Shimen strain was investigated based on the protein sequence. Four mainly hydrophobic regions were identified, spanning residues 8–13, 21–31, 41–46, and 57–66, respectively (Fig. 1B).Fig. 1 Multiple sequence alignment and amphipathic features of classical swine fever virus (CSFV) p7 protein. (A) Residues from isolated CSFV p7 proteins are shown. The alignment is coloured by CLUSTAL. (B) The amphipathicity of CSFV p7 protein by Expasy ProtScale.

Structures of CSFV p7 viroporin predicted by AlphaFold2

The CSFV p7 protein has been demonstrated to form homo-oligomers. The prokaryotic expression system was used to obtain the his-tagged p7 protein, which was shown to exist as a tetramer when treated with glutaraldehyde19. In contrast, the eukaryotic expression of the sumo-tagged p7 protein, incubated with glutaraldehyde, resulted in the formation of pentamers or hexamers20. Therefore, CSFV p7 protein is likely to be a hexameric channel-forming protein as a hexamer acting like HCV p7. Consequently, to better characterize the possible structures of CSFV p7, the tetramer, pentamer, and hexamer were individually obtained by AlphaFold2. The best structures with the highest scores were shown (Fig. 2). Interestingly, the surface of CSFV p7 did not exhibit pore formation in its tetramer or pentamer, whereas the hexamer displayed pore formation, suggesting that CSFV p7 prefers to form channels through hexameric arrangement. The p7 protein encoded by HCV has been known to be a hexamer with ion channel activity within the Flaviviridae family12. Given the similarities within the Flaviviridae family between CSFV p7 and HCV p7, CSFV p7 protein is also likely to assemble in a hexameric arrangement to exhibit ion channel activity. Therefore, further analysis was conducted on the structural characteristics of CSFV p7 hexamer. The results showed that the structure of each monomer consists of four α-helices: Q5-I15 for α-helix I, I20-V32 for α-helix II, E36-M49 for α-helix III, and P53-A68 for α-helix IV (Fig. 3A). Furthermore, the pore channel is mainly constituted by α-helix II, which contains many hydrophobic residues I20, V23, V24, L27, L28, L29, L31 and V32 (Fig. 3B). Residues I20, E21, V24, Y25, L28, L31, V32 and R34 participated the constitution of the pore channel in hexameric assembly (Fig. 3C). Moreover, polar residues Y25 are located inside of the pore channel, potentially facilitating the selective recruitment and dehydration of ions. Furthermore, residues L28 and L31 within the channel possibly act as hydrophobic constrictors, restricting the free movement of water. The hydrophilic and electrically charged residues E21 and R34 were individually positioned at the ends of the channel, suggesting their potential involvement in channel gating (Fig. 3C).Fig. 2 Computational 3D structures of classical swine fever virus (CSFV) p7 protein. The tetramer, pentamer, and hexamer were individually calculated by AlphaFold2 in Google Colab frameworks.

Fig. 3 Overall views of classical swine fever virus (CSFV) p7 hexamer. (A) The structure of the CSFV p7 hexamer. The CSFV p7 hexamer is composed of monomers, and a representation of one monomer is depicted. The monomer is comprised of four α-helices: Q5-I15 for α-helix I, I20-V32 for α-helix II, E36-M49 for α-helix III, and P53-A68 for α-helix IV. The α-helix II, depicted in light blue, formed the ion channel. (B) The surface of the α-helix II was shown. (C) The surface representation of the ion channel was displayed. Residues I20, E21, V23, V24, L27, L28, L29, L31, V32 and R34 collectively contribute to the formation of this ion channel.

Structural validation, motion and stability of CSFV p7 in its tetrameric, pentameric, and hexameric forms

To assess the quality of CSFV p7 structures, we generated Ramachandran plots using PROCHECK (Fig. S1). For the tetramer, 93.9% of the amino acids were in most allowed region, 5.7% were in additional allowed region, and 0.4% were in generously allowed region (Fig. S1A). For the pentamer (Fig. S1B), 92.1% of the residues were in most allowed region, while 6.2% were in additional allowed region, and 1.6% were in generously allowed region. In the case of the hexamer (Fig. S1C), 93.4% of the amino acids were in most allowed region, and 6.6% were in the allowed region.

Molecular dynamics (MD) simulations of 300 ns were performed to validate the 3D structural assemblies and investigate the structural motion and stability of CSFV p7 models (Figs. 4 and 5). Figure 4 indicates that the secondary structures of the tetramer, pentamer, and hexamer were largely preserved throughout the simulations. The stability of CSFV p7 in its different forms was evaluated using the root mean square deviation (RMSD). The tetramer, pentamer and hexamer reached equilibrium after 40 ns, 20 ns and 30 ns respectively and remained stable throughout the trajectory simulation, suggesting that the 3D structural assemblies in different forms were stable (Fig. 5A). These results indicated that the modeled structures are reliable. Additionally, the root mean square fluctuation (RMSF) (Fig. 5B) and radius of gyration (Rg) (Fig. 5C) of the hexamer were higher compared to those of the tetramer and pentamer. This increased variation may indicate conformational changes associated with ion channel activity, suggesting that CSFV p7 likely forms a pore channel in its hexameric assembly.Fig. 4 Analysis of the secondary structure of classical swine fever virus (CSFV) p7 in its tetramer (A), pentamer (B) and hexamer (C) by 300 ns of molecular dynamics (MD) simulations. Right: Structural alignment of the initial structure and snapshot corresponding to 300 ns.

Fig. 5 Structural motion and stability of CSFV p7 in its tetrameric, pentameric, and hexameric forms. (A) Root mean square deviation (RMSD), (B) root mean square fluctuation (RMSF) and (C) the radius of gyration (Rg) values for the backbone at 300 K for last 300 ns.

Analysis of E21A mutants of CSFV p7 in tetrameric, pentameric, and hexameric forms by MD simulations

Previous studies have shown that the introduction of alanine substitutions in residues 17–23 of the CSFV p7 mutant prevents the production of infectious viruses8. Among these residues, we hypothesize that the hydrophilic and electrically charged residue E21 is likely involved in channel gating, based on structural analysis (Fig. 3C). Consequently, we analyzed the E21A mutants of CSFV p7 in different oligomeric forms using MD simulations (Fig. 6). The stability of the mutant hexamer-E21A (Fig. 6A) decreased compared to the wild-type hexamer (Fig. 5A), as indicated by RMSD measurements. In contrast, no significant difference was observed in the mutant tetramer-E21A and pentamer-E21A compared to their wild-type counterparts (Fig. 5), as assessed by several parameters, including RMSD, RMSF, and Rg.Fig. 6 Structural motion and stability of CSFV p7 in its mutants including tetrameric, pentameric, and hexameric forms. (A) Root mean square deviation (RMSD), (B) root mean square fluctuation (RMSF) and (C) the radius of gyration (Rg) values for the backbone at 300 K for last 300 ns.

The structures of CSFV p7-amantadine complex by molecular docking

Amantadine has been shown to inhibit CSFV replication by blocking the activity of its p7 viroporin9,18. To investigate the binding mechanism of amantadine to the CSFV p7, Autodock Vina was employed. After the structures reached equilibrium at 40, 20, and 30 ns during molecular dynamics (MD) simulations, the tetramer, pentamer and hexamer conformations at each of these time points (40, 20, and 30 ns) were individually used for the docking studies. The binding energy from the five best models of the CSFV p7-amantadine complexes were individually used to assess the potential binding regions of amantadine to the CSFV p7 in its tetrameric, pentameric, and hexameric forms. The results showed that amantadine interacts with a predominantly hydrophobic region of the tetramer (Fig. 7A) and pentamer (Fig. 7B), or with the hydrophobic pore channel of the hexamer (Fig. 7C). Notably, the binding regions of amantadine to the hydrophobic pore channel in the hexameric form of CSFV p7 exhibited similarities to the binding regions observed for the M2 ion channel14, suggesting that CSFV p7 may form a pore channel in its hexameric assembly. The RMSD values were observed to be less than 1.459 Å, 1.776 Å, and 0.012 Å for the tetramer, pentamer, and hexamer, respectively (Table 1). The binding affinities were calculated as − 4.6 kcal/mol, − 4.5 kcal/mol, and − 4.8 kcal/mol for the tetramer, pentamer, and hexamer, respectively (Table 1).Fig. 7 Overall views of amantadine with bound classical swine fever virus (CSFV) p7 tetramer (A), pentamer (B) and hexamer (C) by AutoDock Vina, respectively.

Table 1 The docking affinity and the root mean square deviation (RMSD) of amantadine against the CSFV p7 hexamer.

Models	Affinity (Kcal/mol)	Distance from best mode (RMSD)	
Tetramer	1	 − 4.6	0.000	
2	 − 4.6	0.034	
3	 − 4.6	0.006	
4	 − 4.6	1.459	
5	 − 4.6	1.442	
Pentamer	1	 − 4.5	0.000	
2	 − 4.5	0.412	
3	 − 4.5	0.136	
4	 − 4.5	1.562	
5	 − 4.5	1.776	
Hexamer	1	 − 4.8	0.000	
2	 − 4.8	0.011	
3	 − 4.8	0.012	
4	 − 4.8	8.033	
5	 − 4.8	8.084	

Due to the presence of the pore channel formed by the hexamer, the binding sites of amantadine to the hexamer were further analyzed. The binding regions of amantadine were found within the ion channel, involving interaction residues I20, E21, V24 and Y25 (Fig. 8A and B). Furthermore, conventional hydrogen bonds were observed between amantadine and residue E21 by DS Visualizer, while van der Waals forces and Alkyl interactions were established between amantadine and residues I20, E21, V24 and Y25 (Fig. 8C). Considering the symmetrical nature of the hexameric assembly of the CSFV p7 structure, residues I20, E21, V24, Y25 and L28 collectively constitute six druggable regions that are equivalent in nature. This observation implies that these druggable regions have the potential to accommodate up to six amantadine molecules. As a result, models 4 and 5 exhibit higher RMSD values, while still maintaining the same interactions (Table 1 and Fig. 8A).Fig. 8 Analysis of amantadine with bound classical swine fever virus (CSFV) p7 hexamer. (A) The drug amantadine was subjected to molecular docking with CSFV p7 hexamer using AutoDock Vina. Five models were generated and presented as protein surfaces. (B) The residues of CSFV p7 that interact with amantadine were displayed using a ribbon representation, highlighting the specific regions of interaction. (C) 2D interactions diagrams between amantadine and the residues of CSFV p7.

Structural motion and stability of CSFV p7-amantadine complex by MD simulations

To further analyze the dynamic behavior of amantadine within the druggable regions of CSFV p7 tetramer, pentamer, and hexamer, a series of 300 ns MD simulations were conducted on the CSFV p7-amantadine complexes. The trajectory of the CSFV p7-amantadine was analyzed via RMSD, RMSF and Rg values of their backbone atoms. The RMSD analysis revealed that the CSFV p7 hexamer-amantadine complex reached equilibrium after 40 ns, similar to the wild-type CSFV p7 hexamer, and stabilized at 0.5 nm, which is higher compared to the wild-type CSFV p7 hexamer (Fig. 9A). This suggests that amantadine binding may induce structural changes in the hexamer. In contrast, the RMSD values for the CSFV p7 tetramer and pentamer, as well as their respective complexes with amantadine, did not show significant differences, indicating that amantadine binding does not cause structural alterations in the tetramer and pentamer forms. The RMSF analysis demonstrated a decrease in the CSFV p7 hexamer’s RMSF following amantadine binding, reflecting a stabilization of the structure (Fig. 9B). No significant changes in RMSF were observed for the CSFV p7 tetramer and pentamer after binding with amantadine. Additionally, the Rg values for the CSFV p7 tetramer, pentamer, and hexamer showed no significant changes upon amantadine binding (Fig. 9C). Furthermore, the hexamer-amantadine complex exhibited a higher number of hydrogen bonds in non-covalent interactions compared to the tetramer-amantadine and pentamer-amantadine complexes (Fig. 9D).Fig. 9 Trajectory analysis of molecular dynamics (MD) simulations of classical swine fever virus (CSFV) p7-amantadine complexes. (A) Root mean square deviation (RMSD), (B) root mean square fluctuation (RMSF) and (C) the radius of gyration (Rg), values for the backbone at 300 K for last 300 ns. (D) Intramolecular hydrogen bonds in the complexes of the amantadine-CSFV p7 complexes in tetrameric, pentameric, and hexameric forms, respectively.

Principal component analysis (PCA) of CSFV p7 in its wild types, mutants and complexes with amantadine

PCA was performed to support the results of the MD simulation and to understand the structural and conformational changes of CSFV p7 in its wild types, mutants, and complexes with amantadine by calculating the atomic fluctuation covariance matrix (Fig. 10A,C,E). The projection of PC1 and PC2 showed that the mutant hexamer-E21A (Fig. 10D) presented reduced displacement and covered a narrower spatial range compared to the wild-type hexamer (Fig. 10B). This suggests that the ion channel activity may be diminished due to the E21A mutation. Moreover, the binding of amantadine to the hexamer resulted in the most restricted movement (Fig. 10F), indicating that amantadine may block the ion channel activity by stabilizing the structural conformation. In contrast, the tetramer-E21A and pentamer-E21A mutants showed less change in displacement (Fig. 10D). Additionally, amantadine binding to the tetramer resulted in more significant displacement and a wider spatial range, which is inconsistent with current research and suggests that CSFV p7 may not exist as a tetramer. Collectively, these results potentially indicate that CSFV p7 may form a pore channel primarily in its hexameric assembly.Fig. 10 Principal components analysis of classical swine fever virus (CSFV) p7, its mutants and complexes with amantadine. (A) Eigenvectors of the covariance matrix and (B) projection of the movement in the phase space between the first and second eigenvectors (PC1 vs. PC2) of CSFV p7 in its in tetrameric, pentameric, and hexameric forms. (C) Eigenvectors of the covariance matrix and (D) projection of the movement in the phase space between the PC1 vs. PC2 of CSFV p7 in its mutants including tetramer-E21A, pentamer-E21A and hexamer-E21A. (E) Eigenvectors of the covariance matrix and (F) projection of the movement in the phase space between PC1 vs. PC2 of CSFV p7 in its complexes including tetramer-amantadine, pentamer-amantadine and hexamer-amantadine.

Possible molecular model for ion transport of the pore channel formed by hexamer and its inhibition by amantadine

The typical configuration of a gated ion channel consists of pore elements and a gating mechanism. The pore elements facilitate the selectivity of specific ions, while the gating mechanism transiently opens the channel to allow ion permeation. Based on the possible hexameric assembly of CSFV p7 structure, along with mutant analysis, docking studies, and MD simulations, we proposed a model of CSFV p7 channel as a gated ion channel (Fig. 11). Within this model, residues E21, Y25 and R34 are suggested to selectively recruit and dehydrate ions, whereas residues L28 and L31 restrict the free movement of water as hydrophobic constrictors. By binding to residues I20, E21, V24 and Y25, amantadine effectively interferes with ion transport by blocking the channel activity. However, this proposed molecular model requires experimental validation.Fig. 11 A proposed model for the inhibition of the classical swine fever virus (CSFV) p7 channel by the amantadine. Amantadine blocks the channel by binding the residues I20, E21, V24 and Y25 that may be required for ion transportation and channel opening.

Discussion

Machine learning and computational methods played a significant role in structural analysis and revolutionizing drug discovery. AlphaFold2 showed remarkable accuracy21. Using AlphaFold2 and clustering, new deaminases were identified to investigate unknown deaminase properties such as efficiently editing cytosine bases22. Combining with an industry-leading molecular docking method, proteins predicted by AlphaFold2 were explored for structure-based virtual screening23. Moreover, the RoseTTAFold algorithm was employed to undertake de novo protein structure design, enabling the development of protein binders, symmetric oligomer design, and scaffolding of enzyme active sites24. These advancements highlight the immense potential of machine learning and computational methods in hit identification and drug discovery, primarily based on protein structure prediction. Building upon these facts, our study employed AlphaFold2 and molecular docking to elucidate the molecular mechanisms by which amantadine targets the CSFV p7 ion channel. However, it is essential to acknowledge that our findings are preliminary and require experimental validation.

CSFV and HCV belong to the family Flaviviridae. In the Protein Data Bank, HCV p7 structure (PDB: 2m6x) was reported as a hexametric assembly. Consequently, we used HCV p7 structure as a reference template for homology modeling of the CSFV p7 structure by AlphaFold2, despite a sequence alignment showing only 28.26% similarity between CSFV p7 and HCV p7 (Fig. S2). However, AlphaFold2 did not utilize the HCV p7 model during the structural prediction process due to the substantial dissimilarity between the CSFV p7 sequence and the HCV p7 model. We therefore had to use other methods to validate the CSFV p7 structure. Firstly, to validated 2D structures of CSFV p7, CSFV p7 structures firstly were validated by Ramachandran plots and molecular dynamics (MD) simulations (Fig. S1 and Fig. 4). For the validation of 3D structural assembly, several parameters of MD simulations were evaluated to validate stability of the structural assembly in tetramer, pentamer, or hexamer (Fig. 5). In addition, to validate the predicted structures, two webserver tools, namely SAVESv6.0 and PAE Viewer, were employed to facilitate the display of structure validation. However, the outcomes from SAVESv6.0 and PAE Viewer indicated comparatively lower evaluation scores for the HCV p7 model used as a control (data not shown). This indicates a potential limitation of these tools in assessing the structures of homomultimeric proteins.

CSFV p7 plays a crucial role in the generation of viral particles. Constructs lacking specific regions in CSFV p7, namely CSFV p7Δ10–32 and CSFV p7Δ15–51, consistently fail to yield viable infectious particles8. Furthermore, the introduction of alanine substitutions in p7 residues 17–23 of the CSFV mutant prevents the production of infectious viruses8. Our computational analysis conducted in this study elucidates that the residue E21 in CSFV p7 is accountable for the activity of ion channels, thereby corroborating these findings. Additionally, it has been observed that the fragment of CSFV p7 encompassing residues 33–67 has been identified to be important for the ion channels activity9. Our computational analysis in this study confirms the involvement of the residue R34 in CSFV p7 in the activity of ion channels, thus reinforcing the consistency with previous results. Consequently, these results significantly enhance the credibility and robustness of our computational findings.

Several viroporins mediate ion transport by forming pH-gated or H+ conducting channels, depending on the pH change in the environment6. However, the exact mechanism of ion transport from into and out of the cell is still unclear. Some progress has been made on IAV M2 and HCV p7. In the case of HCV p7, it has been proposed that residues I6 and N9 rings are the critical region for channel gating. The N9 ring is believed to contribute to ion selectivity by recruiting and dehydrating ions near the channel’s exit, while the I6 ring acts as a hydrophobic constrictor restraining the water movement of freely passing through12. As for IAV M2, it has been hypothesized that pH-dependent interactions between H37 and W41 play a crucial role in the channel opening process. Specifically, it has been proposed that the H37 imidazole ring forms a cyclic hydrogen bond that closes the proton channel, while disruption of this bond at low pH allows the channel opening via the interaction of the W41 indole ring with H37. In this study, we investigated the residues involved in the construction of the ion channel in CSFV p7, which include residues I20, E21, V24, Y25, L28, L31, V32 and R34. Among these residues, E21 and R34, located at the entrance and exit of the channel, possess electrical charges and likely participate in ion recruitment and release via a pH-dependent mechanism involving structural rearrangements. Within the channel, the polar nature of Y25 within the ion channel suggests that it may facilitate ion transport, while the hydrophobic properties of residues L28 and L31 imply the limitations on water movement. Based on the characteristics of a gated ion channel and the analysis of the constituent residues in the CSFV p7 channel, we propose a potential mechanism for the ion transport of CSFV p7. Combing the docking studies, we further propose that the binding of amantadine inhibits channel activity by restricting structural rearrangement.

Conclusion

This study provides a potential structural model and molecular explanation for the ion channel conductance mediated by CSFV p7, as well as the inhibitory by amantadine. CSFV p7 may oligomerize to form a pore channel in hexameric assembly. Residues Y25, L28 and L31 likely contribute to channel activity, including ion transport and water movement, while residues E21 and R34 potentially involve in the gating mechanisms of the channel. Binding of amantadine to residues I20, E21, V24 and Y25 effectively blocks the channel activity. However, this proposed molecular model requires experimental validation.

Materials and methods

Structural calculation of CSFV p7 by AlphaFold2

The prediction of CSFV p7 protein structures was conducted on the AlphaFold2 pipeline within the Google Colab21. The query sequence of CSFV p7 utilized in this prediction is from CSFV Shimen strain, which is available on GenBank under accession number AF092448.2. During the prediction process, the model type selected was alphafold2_multimer_v3, with the templated mode set to none. The MSA mode utilized MMseqs2 in combination with the UniRef + Environmental database. Both unpaired and paired pairing modes were specified. A total of five models were generated through 48 cycles of interactive prediction. Finally, the best model with the highest score was used for the further analysis in this study.

Molecular docking for CSFV p7 with amantadine

Input files for rigid protein and flexible ligands were prepared using the AutoDock Tools 1.5.625. Polar and nonpolar hydrogen atoms were added and merged individually to the protein structure. Kollman charges and solvation parameters were determined by default settings. Gasteiger charges were added to the minimized ligand structures, and all bonds were made rotatable and flexible by allowing the detection of root torsion. The CHARMM energies were estimated on a grid defined by the following parameters: center_x = 0.895, center_y = 0.005, center_z = 6.278, size_x = 24.75, size_y = 24.75, and size_z = 24.75. The grid box was set to 30 Å × 30 Å × 30 Å, with a grid point distance of 0.05 nm. For docking calculations, AutoDock Vina was employed. The output was subsequently clustered based on the RMSD. In total, five binding modes were generated.

Molecular dynamics simulations

Molecular dynamics simulations of the CSFV p7 in its tetramer, pentamer and hexamer, its mutants, and complexes with amantadine were individually performed by GROMACS over a period of 300 ns26. The protein topologies were generated by the CHARMM36 all-atom force field, while the ligand topologies were generated using the CHARMM force field. The complexes were formed by combining the ligand and protein topologies for each specific case being studied. Following this, the complex was immersed in a cubic box with dimensions of 1.0 nm and solvated with the transferable intermolecular potential with 3 points (TIP3P) water model. The system was then neutralized using Na and Cl ions. To optimize the energy, an energy minimization step was carried out using the steepest descent algorithm for 20,000 steps. Subsequently, the equilibration was performed in separate 100 ps intervals. Finally, a molecular dynamic simulation was performed with a time step of 2 fs, employing the Particle Mesh Ewald (PME) method. Extended molecular dynamic runs were carried out for 300 ns. The dynamics data were calculated by the GROMACS tools. The secondary structure calculation was carried out in the VMD program using the timeline plug-in through the entire trajectory simulation of 300 ns.

Principal component analysis

The goal of protein simulation is to generate enough configurations of the system of interest to identify functionally relevant movements. Principal component analysis (PCA) has been widely used to reduce the complexity of the data obtained from MD simulation trajectories by recovering the collective movement of atoms in the simulated trajectories that are essential for biological processes. PCA was performed with the data extracted from a 300 ns MD simulations. The projected eigenvectors and eigenvalues obtained during the first two principal components (PC1 and PC2) runs were calculated with the GROMACS tools.

Supplementary Information

Supplementary Figure S1.

Supplementary Figure S2.

Supplementary Legends.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71477-1.

Author contributions

XC and XW collected the data. XC and XW wrote the manuscript and draw all the figures. All authors contributed to the article and approved the submitted version.

Funding

This study was supported by Binzhou Medical University.

Data availability

The original data and materials presented in the study are included in the article.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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