Conservation and hydrophobicity of CSFV p7 proteinThe 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. 1Multiple 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 AlphaFold2The 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. 2Computational 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. 3Overall 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 formsTo 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. 4Analysis 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. 5Structural 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 simulationsPrevious 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. 6Structural 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 dockingAmantadine 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. 7Overall 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.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. 8Analysis 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 simulationsTo 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. 9Trajectory 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 amantadinePCA 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. 10Principal 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 amantadineThe 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. 11A 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.
