Restraint of VP1 Protein of Foot and Mouth Disease Virus using Specific Antiviral Peptides: an in Silico Investigation

1. Introduction

Foot and mouth disease (FMD) is caused by a virus (FMD virus) that contains positive-strand ribonucleic acid (RNA) and belongs to the genus Aphthovirus and Picornaviridae family ( 1 ). The FMD has highly variable RNA that engenders O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3 serotypes and a wide range of subtypes. Regarding the distribution of seven FMD virus serotypes worldwide, serotypes A and O have the highest distribution rates. They are the most antigenically variable European-Asian serotypes and are also important serotypes in the Middle East and especially in Iran ( 2 ). The FMD virus contains an icosahedral protein capsid (containing 20 equilateral triangles) and an RNA genome that encodes a polyprotein with four structural proteins (including VP1, VP2, VP3, and VP4) and eight non-structural proteins (including L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D polymerase) ( 3 , 4 ).

The capsid of the FMD virus consists of 60 structural units called protomers, and each protomer consists of 1AB, 1C, and 1D proteins ( 5 ). The X-ray crystallography of the VP1 protein has confirmed that the tertiary structure of this protein is in the beta-sheet form which can create some loops on the surface of the virus. In general, the G-H loop with highly variable regions is known as the most important loop of VP1 protein, including 140-160 residues of VP1 protein. In fact, the flexibility of the G-H loop plays a key role in the binding and entry of the virus into the host cell. It has been reported that the binding and entry of the virus into the host cell completely decreases while the G-H loop is located on the viral cover due to the deformation of disulfide bonds ( 6 , 7 ). The G-H loop has a protected region containing arginine (Arg), glycine (Gly), and aspartic acid (Asp) amino acids which are located in 145, 146, and 147 positions, respectively. These amino acids play a vital role in binding the virus to the host cell receptor ( 8 ).

Antimicrobial peptides (AMPs) are small biological molecules that play an important role in the defense mechanisms of organisms by invading microorganisms, maintaining gastrointestinal homeostasis, and modulating inflammatory responses. These compounds are found in both eukaryotes and prokaryotes which exhibit a wide variety of antimicrobial activities (antibacterial, antiviral, and antifungal) ( 9 ). In general, these peptides are divided into two categories in terms of charge, namely cationic and anionic.

Moreover, they are divided into four groups in terms of their secondary structure, including α-helix peptides, β-plates, fast or helical peptides, and finally elongated peptides. The α-helical peptides (e.g., scorpions, meganins, and LL37) and β-platelets (e.g., human alpha and beta-defensin, pleistacins, and protogrins) are known as the most frequent AMPs in the world ( 10 ). In addition, some peptides do not belong to any of these groups and form their active structure when they interact with the target cell membrane or form a different structure in aqueous or fatty media ( 9 ).

It has been reported that peptides can also be effective against viral infections ( 11 , 12 ). Many studies have reported that some peptides could exhibit strong antiviral activities in both in vitro and in vivo conditions while in contact with the virus. These peptides can kill viruses in a variety of ways, including inactivation of the virus, prevention of the virus from entering cells, interaction with specific cell receptors, or by blockage of viral proteins that target cells ( 12 , 13 ). Hence, it seems that the function of the FMD virus can be disrupted by the inhibition of VP1 protein via proper antiviral peptides. In this regard, the present study aimed to select appropriate anti-FMD virus peptides among potential antiviral peptides to block the VP1 protein of the FMD virus.

2. Materials and Methods

2.1. Data collection and antimicrobial possibility investigation

To collect amino acid sequences of potential antiviral peptides, the CAMPR3 database (http://www.camp.bicnirrh.res.in/) was employed. It must be mentioned that the collection was performed based on length (1-100 mer), experimental validity, and antiviral quality. Moreover, the VP1 gene sequence of serotype O virus isolated in Iran in 2010 (FMDV O/2010/IRN) was obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/) with HQ663879 accession number. The amino acid sequence of VP1 protein was artificially obtained by CLC Main Workbench software (version 5.5).

To investigate the AMP probability of the potential antiviral peptides, the Antimicrobial Peptide Scanner vr.2 server (https://www.dveltri.com/ascan/v2/ascan.html) was applied with a threshold > 0.5. In this case, the amino acid sequence of each potential antiviral peptide was submitted in FASTA format. Moreover, to increase the sensitivity of the selection, the potential antiviral peptides with a score higher than 0.7 were selected. Finally, the remaining potential antiviral peptides were employed to the next analysis.

2.2 Evaluation of physicochemical features

In the current study, the most important physicochemical features of the remaining potential antiviral peptides, such as pI, GRAVY, instability index, and net charge, were investigated by the ProtParam server (https://web.expasy.org/protparam/). To predict these parameters, the raw amino acid sequence of each peptide was pasted on the server. The peptides whose instability index was more than 40 or loaded with negative or zero net charges were eliminated from the subsequent analysis. Afterward, the hydrophobicity/hydrophilicity parameter was assessed by PEPTIDE 2.0 server (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php) to filter the remaining potential antiviral peptides from the previous step. Finally, the Protein-Sol server (https://protein-sol.manchester.ac.uk/) was employed to evaluate the solubility parameter using the FASTA format of the amino acid sequence of each remaining antiviral peptide.

2.3. Prediction of the tertiary structure

The remained antiviral peptides, which had more than 30% hydrophobic amino acid residues in their structure along with VP1 protein, were modeled using the I-TASSER server (https://zhanggroup.org/I-TASSER/). The best model of VP1 protein by this server, which had the highest confidence score (C-Score), was refined and then validated using GalaxyRefine (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE2) and Volume, Area, Dihedral Angle Reporter (http://vadar.wishartlab.com/) servers, respectively.

2.4. Molecular docking

The interaction between the VP1 protein as receptor and the selected antiviral peptides as ligands was separately performed using the ClusPro 2.0 server (https://cluspro.bu.edu/login.php). The ClusPro is a fully automated server used to predict protein-protein interaction. In the first phase, the server used Piper, an FFT-based hard docking program, to create 1,000 low-connection complexes. In the next step, the server categorized the predicted connections and removed those that were more unstable. Finally, the selected complexes were optimized by Monte Carlo simulation ( 14 ). Finally, the hydrogen bonds resulting from this interaction were investigated and visualized by LigPlot + and PyMol software.

3. Results

3.1. Data collection and antimicrobial possibility investigation

As mentioned earlier, in the current study, primary antiviral peptide collection was conducted based on length (1-100 mer), experimental validity, and antiviral quality. According to these features, 28 potential antiviral peptides with a length of 25-43 amino acids were selected to be evaluated regarding their ability to inhibit the VP1 protein of the FMD virus (Table 1). Results of the antimicrobial possibility investigation revealed that all potential antiviral peptides had high probability values (Table 1).

3.2. Assessment of the physicochemical parameters

In the current project, based on the physicochemical parameters predicted by ProtParam online server, Circulin-D, Circulin-E, Caerin-1.1, Vhl-2, Neutrophil defensin 3, Defensin-6, Mytilin-B, Melittin, Vhl-1, Palicourein, Corticostatin-6, Corticostatin-3 precursor, Corticostatin-4 precursor, and Human Defensin-5 peptides were removed from further analysis due to their negative or zero net charge or high instability index (>40) (Table 2). Among the remaining potential antiviral peptides, the highest length, and consequently, the highest molecular weight belonged to the Ginkbilobin peptide, while the lowest weight belonged to Maximin 1 and Cycloviolin-B peptides. Moreover, the highest observed pI was related to the Ginkbilobin peptide. The highest and the lowest GRAVY parameters were -0.608 and 1.188 which belonged to Reptilian Defensin and Caerin-1.1 peptides, respectively (Table 2).

3.3. Hydrophobicity and solubility

As it is summarized in table 3, the Protein-Sol server determined that all the remaining peptides from the previous step were soluble (Table 3). Hydrophobicity of the selected peptides from the previous step was successfully determined using the PEPTIDE 2.0 server. As reported in table 3, the selected peptides were sorted according to their number of hydrophobic amino acids (%). The results demonstrated that the Dermaseptin-3 peptide with 53.33% of hydrophobic amino acid residues contained the highest number of hydrophobic amino acids, while the Kalata-B8 peptide with 16.13% had the lowest number of hydrophobic amino acid residues (Table 3).

3.4. Determination and Validation of Tertiary Structures

Tertiary structures of Dermaseptin-3, Ginkbilobin, Circulin-F, Maximin 1, Cycloviolin-A, Cycloviolin-D, Circulin-C, and Cycloviolin-C and Antihypertensive protein BDS-1 peptides (as final antiviral peptides), as well as VP1 protein, were modeled with -0.43, -1.41, 0.31, -0.60, 0.61, 0.64, 0.87, 0.68, -0.53 and 1.30 C-score, respectively. In the next step, to achieve the most accurate folded state of the presented VP1 model, the GalaxyRefine server was employed to refine the best model and then was verified using Ramachandran analysis. In the refined model of VP1 protein, 93%, 5%, 0%, and 0% of the residues were located in the core, allowed, generous, and outside regions, respectively, while these indexes in the primary model predicted by the I-TASSER server were 74%, 22%, 3%, and 0%, respectively (Figure 1).

Figure 1.A: tertiary structure of the VP1 protein modeled by I-TESSAR server. B: Ramachandran plot of the primary model of VP1 protein. In this model, 74%, 22%, 3% and 0% of the residues were located in core, allowed, generous and outside regions, respectively. C: Ramachandran plot of the refined model of VP1 protein. In this model, 93%, 5%, 0% and 0% of the residues were located in core, allowed, generous and outside regions, respectively

3.5. Molecular docking

Investigations were conducted to examine the possible interaction between the VP1 protein and final antiviral peptides with more than 30% hydrophobic amino acid residues content (including Dermaseptin-3, Ginkbilobin, Circulin-F, Maximin 1, Cycloviolin-A, Cycloviolin-D, Circulin-C, Cycloviolin-C, and Antihypertensive protein BDS-1 peptides) using the ClusPro 2.0 server. Results of molecular docking showed that the lowest energy levels of docking between VP1 protein and Dermaseptin-3, Ginkbilobin, Circulin-F, Maximin 1, Cycloviolin-A, Cycloviolin-D, Circulin-C, Cycloviolin-C, and Antihypertensive protein BDS-1 peptides were -743.4, -1295.4, -856.3, -715.2, -866.2, -865.0, -814.4, -864.0, and -951.9 kcal/mol, respectively.

Further analysis was performed to determine the number and length of hydrogen bonds using LigPlot+ software. Based on the results, among the mentioned peptides, Dermaseptin-3 (Figure 2) and Ginkbilobin peptides (Figure 3) formed the highest number of hydrogen bonds (10 bonds) with nine and seven amino acid residues from VP1 protein, respectively (Table 4). In the next rank, Cycloviolin-A and Circulin-C peptides were also able to establish nine hydrogen bonds with eight and seven amino acids of VP1 protein, respectively. In addition, Circulin-F and Maximin I peptides with two and three hydrogen bonds showed the lowest number of bindings with VP1 protein, respectively. (Table 4).

Figure 2. (A1) Interaction between Dermaseptin-3 peptide (the green color) and VP1 protein (the blue color) which was created by ClusPro server and visualized by PyMol software. (A2) The exact docked region which was visualized by PyMol software (the blue color refers to amino acid residues which take part in hydrogen bond formation and the red color represents the hydrogen bonds). (A3) The docked regions which were analyzed by LigPlot + software (the green amino acids (B) belong to Dermaseptin-3 peptide, the blue amino acids (A) belong to VP1 protein and the green dotted lines represent hydrogen bonds)

Figure 3. (A1) A scheme of docking the Ginkbilobin peptide (the green color) and VP1 protein (the blue color) which was visualized by PyMol software. (A2) The region for establishing hydrogen bonds between two domains which was visualized by PyMol software (the blue color is related to the amino acids that participated in the establishment of the hydrogen bond and the red color of the hydrogen bonds). (A3) A scheme of hydrogen bonds (the green dotted line) between the amino acids of the Ginkbilobin peptide (the green amino acids (B)) and VP1 protein (the blue amino acids (A)) which was analyzed by LigPlot + software

4. Discussion

Bioinformatics is an interdisciplinary science that includes methods and software used for understanding biological information. The increasing volume of genomic data and the need to store, retrieve, and properly analyze this data has led to the emergence of bioinformatics ( 15 ). Today, this knowledge, as a powerful science, leads to the production of highly accurate data, at a much lower cost, compared to an in vitro study. In fact, bioinformatics analyzes biological data that is used in various fields of biology, such as drug discovery and vaccine design ( 16 ). In this regard, the study of the structure and activity of macromolecules in different conditions and also interactions between them is necessary to better understand cellular function at the molecular level in the biological sciences. ( 17 ). Therefore, as a method based on the investigation of the structure of the protein-protein complex in drug design, molecular docking is a key tool in the structural molecular biology and computational design of drugs.

The purpose of ligand-protein binding is to predict the state or states of the major ligand binding to a protein with a known three-dimensional structure ( 18 ). Today, the application of such bioinformatic techniques in virtual screening or de-novo design, before the preparation and synthesis of biomolecules in in vitro studies, has received a lot of attention from researchers as it allows them to enter the experimental phase of research with a valuable perspective of the function of peptides and proteins. ( 19 - 21 ).

Usage of antibiotics is usually the first option in disease treatment with a microbial origin; however, today, the issue of antibiotic resistance is very worrying. Microorganisms can show resistance to antibiotics using several methods, including the following: (1) limiting the use of antibiotics by microorganisms through the reduction of their permeability and alteration of their hydrophobic properties ( 22 ); (2) changing the goals of drugs when antibiotics target multiple sites in a cell ( 23 ); (3) inactivating antibiotics through the enzymatic mechanism of microorganisms ( 24 ); and (4) overexpressing transporter genes and efflux pumps that divert the path of the antibiotic to its target position in the microorganism ( 25 ).

Therefore, antibiotic resistance is one of the most important challenges of the treatment. The AMPs are natural oligo-peptides that belong to inherently immune components of organisms and have significant action against pathogens. Due to their significant antibacterial and antiviral activities, it has recently been reported that AMPs have received more attention as a safer alternative for the treatment of diseases caused by bacteria or viruses ( 26 ). Until now, the importance of using AMPs as a safe treatment with the least side effects has been proven in many studies, such as using Mycobacterium leprae in human skin diseases in Leprosy disease, pig cathelicidin PR-39 in skin lesions, and Staphylococcus aureus in atopic dermatitis ( 27 ).

In another study, researchers have proved that a synthesized form of the Epinephelus coioides antimicrobial peptide, epinecidin-1 (Epi-1), could combat FMDV. The Epi-1 is known to have broad-spectrum antimicrobial activity and low toxicity toward normal eukaryotic cells, making it a good candidate for use as a therapeutic agent ( 28 ). The FMD is one of the most contagious viral diseases among livestock, which ranks first in the list of infectious diseases of animals.

Despite low mortality rates, it has been reported that FMD drastically reduces livestock production and imposes significant trade restrictions on livestock and livestock products ( 29 , 30 ). The FMD (FMDV, family Picornaviridae, genus Aphthovirus), is structurally distinct from other members of its family due to the presence of a G-H-protruding loop from the VP1 protein. The G-H loop is a motif consisting of 20 amino acid residues in the range 140-160 ( 31 ). Based on the results of this research, Cycloviolin-A, Dermaseptin-3, Ginkbilobin, and Circulin-C peptides with 9, 10, 10, and 9 hydrogen bonds, respectively, showed the strongest bonds with the VP1 protein.

Among these peptides, Cycloviolin-A peptide, through its numerous hydrogen bonds, has been able to inhibit the Asn 143 and Arg 140 amino acid residues of the protein and VP1 by hydrogen bonding with a length of 3.22 Aº and 2.81 Aº, respectively. These amino acids are located in the G-H loop (in the range of 140-160 amino acid residuals), which seems to have a more favorable effect on the inhibition of VP1 protein binding to the cell wall. Therefore, based on the reported results in the present study, it can be concluded that the Cycloviolin-A, Dermaseptin-3, Ginkbilobin, and Circulin-C peptides could be suitable and promising candidates for the drug design to combat the dangerous FMD disease.

The current study was designed to identify specific anti-FMD virus peptides. In this case, 28 potential antiviral peptides were collected from the database and the most important features of these peptides were investigated by reliable servers and software. The results of this study revealed that Dermaseptin-3 and Ginkbilobin peptides were able to strongly inhibit the VP1 protein using 10 hydrogen bonds. Therefore, these peptides were introduced as promising candidates to prevent the FMD virus.

Authors' Contribution

The project was designed by Ali Forouharmehr, whereas Narges Nazifi and Amin Jaydari conducted the different analyses.

Ethics

This article does not consist of any studies with animals performed by any of the authors.

Conflict of Interest

The authors declare there is no conflict of interest.

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