Structural insights into the RNA interaction with Yam bean Mosaic virus (coat protein) from Pachyrhizus erosus using bioinformatics approach.

Plants are constantly threatened by a virus infection, i.e., Potyviruses, the second largest genus of plant viruses which results in several million-dollar losses in various essential crops globally. Yam bean (Pachyrhizus erosus) is considered to be one of the essential tuberous legume crops holding a great potential source of starch. Yam Bean Mosaic Virus (YBMV) of Potyvirus group belonging to the family potyviridae affects Yam bean and several angiosperms both in the tropical and sub-tropical regions causing large economical losses in crops. In this study, we attempted to understand the sequence-structure relationship and mode of RNA binding mechanism in YBMV CP using in silico integrative modeling and all-atoms molecular dynamics (MD) simulations. The assembly of coat protein (CP) subunits from YBMV and the plausible mode of RNA binding were compared with the experimental structure of CP from Watermelon mosaic virus potyvirus (5ODV). The transmembrane helix region is present in the YBMV CP sequence ranging from 76 to 91 amino acids. Like the close structural-homolog, 24 CPs monomeric sub-units formed YBMV a conserved fold. Our computational study showed that ARG124, ARG155, and TYR151 orient towards the inner side of the virion, while, THR122, GLN125, SER92, ASP94 reside towards the outer side of the virion. Despite sharing very low sequence similarity with CPs from other plant viruses, the strongly conserved residues Ser, Arg, and Asp within the RNA binding pocket of YBMV CP indicate the presence of a highly conserved RNA binding site in CPs from different families. Using several bioinformatics tools and comprehensive analysis from MD simulation, our study has provided novel insights into the RNA binding mechanism in YBMV CP. Thus, we anticipate that our findings from this study will be useful for the development of new therapeutic agents against the pathogen, paving the way for researchers to better control this destructive plant virus.

Introduction

Yam bean (Pachyrhizus erosus), also known as “Jicama” belongs to the family Fabaceae is an economically high-yielding leguminous tuber crop showing the widest adaptation with an enhanced nutritional value [1]. It is cultivated broadly in Mexico, Singapore, China, the Philippines, Indonesia, Hawaii, and India. It is the native crop of Tropical America, some parts of Africa, and Asia. In India, it is cultivated in many regions of Assam, Orissa, Bihar, Tripura and West Bengal [2]. Yam bean mosaic virus was discovered for the first time in Brazil [3]. Yam bean (Pachyrrhizus erosus) is also one of the well-known legumes known as “Bengkuang” widely found in Indonesia. It is cultivated mainly in several regions of West and Central Java in Indonesia [4]. Infected plants typically exhibit leaf green vein banding, moulting, curling, inter-veinal mosaic, stunted development and the disease has been known to cause severe production losses in yam [5, 6]. The virus is distributed non-persistently through aphids and vegetative propagation. YMV has been identified in various Dioscoreaceae plants [7-10] and can be mechanically transferred to Chenopodium amanticolor, Nicotiana megalosiphon and Nicotiana benthamiana [8]. Yam bean serves a key function in delivering food and protein-rich micronutrients and protein for human use [10]. The subtribe Glycininae belongs to the genus Pachyrhizus which consists of an essential legume crop named soybean (Glycine max) [11, 12]. The genus involves three cultivated species i.e., Pachyrhizus tuberosus, Pachyrhizus erosus, and Pachyrhizus ahipa produce roots with high storage capacity as well as with high proteins i.e. up to 12% in dry matter, iron content (up to 52 ppm) followed by zinc (up to 14 ppm) [13]. The genomic size of the yam bean is estimated to be between 572 and 597Mbp [14]. The most widely consumed yam bean species are tuberosus and erosus, which have different morphologies within the species and genus [15]. Viruses, on the other hand, are becoming a serious restriction for tuber crops and roots, particularly in the tropics [13].

Sorensen and co-authors reported that 20–40% of yield declination is due to certain viral diseases in Pachyrhizus erosus [13]. Despite of the negative impacts on yam bean crops, there is very little information available on viral diseases. The Yam bean (P. erosus) usually gets infected by bacteria like Pseudomonas [16], fungi [17, 18], insect-pests (i.e., aphids, mealybug, and bruchids) [13], nematodes and viruses. It has been earlier reported that there are two major viruses destroying yam bean cultivation i.e., bean common mosaic virus (BCMV) and sincama mosaic virus (SMV) [13]. Damayanti and colleagues identified a strain causing massive damage to Yam Bean (Pachyrhizus erosus) cultivation in different parts of Indonesia and recognized it as BCMV. Latter the virus was observed to be a distinct strain and named as BCMV-IYbn. Soon after in Peru, certain unique strains were observed that were suspected to be BCMV. Following that, a virus with a distinct strain was obtained for observation, and tests were performed to identify it [19]. Small interfering RNA (siRNA) sequencing took place and assembly was performed on a sample resulting in severe mosaic and leaf deformation [20]. Finally, after the analysis, it was proved that the virus was not BCMV and later Fuentes and co-authors in 2012 proposed the name of the new virus as Yam bean Mosaic Virus (YBMV). The YBMV CP comes under the Potyviridae family consisting of virions i.e., non-enveloped as well as flexuous and filamentous. On the other hand, certain phylogenetic analyses were performed using only the CP (coat protein) of YBMV from the isolates of Vietnam [21] and Indonesia [19] and found maximum identity to YBMV over the CP encoding region which assures the presence of YBMV in the virus as identified as BCMV-IYbn previously. In Peru, Indonesia, and Vietnam, the presence of YBMV and associated viruses in a yard-long bean, yam bean, and black bean respectively suggests the presence of this virus in geographically remote areas and various crops [22]. The potyviral CP is a multifunctional protein that plays a part in almost every phase of the virus life cycle, in addition to its structural purpose of preserving the viral DNA. Interactions between the viral RNA and CP, in particular, must be closely regulated for the RNA to be assigned to each of its roles correctly during the infection process [23].

Cucumber mosaic virus, infection causes mosaic symptoms in a wide range of agricultural plants, including cucumbers. The replacement of amino acid 129 in the coat protein of the Cucumber mosaic virus results in structural alterations in the virus [24]. The screening of potential inhibitors against the coat protein of the Apple Chlorotic Leaf Spot Virus was carried out. Apple chlorotic leaf spot virus (ACLSV) coat protein (CP) is an important latent virus on the apple plant that has been identified [25]. The coat protein (CP) of the tobacco mosaic virus (TMV) self-assembles in transgenic plants that have been deprived of viral RNA, forming aggregates that are dependent on the physical parameters of the environment [26]. A biosimulation technique was used to identify plant-based analogs that might serve as potential inhibitors against the tobacco mosaic virus [27]. siRNAs created by the plant’s defensive system, the host RNA silencing mechanism, target the viral RNA or DNA and function in post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) pathways, respectively, to inhibit viral replication. Host gene expression modulation by virus- and virus-derived small RNAs: new insights into the pathogenesis of viruses and viruses-derived small RNAs [28]. Because of its critical function in viral replication, the RNA-dependent RNA polymerase (RdRp) has emerged as a potential therapeutic target [29].

Previously, there was no protein-related information in the yam bean crop and thus there has been no work done on functional and structural protein characterization of YBMV globally. In 1988, Shukla and Ward investigated the structural characterization of the potyvirus (coat proteins) CP and observed that the amino acid sequences of CP are sufficient to identify and differentiate the individual potyvirus and its strains [30-32]. The coat protein is considered to be the major gene product in the virion [33]. The genome of potyvirus consists of eight different gene products of which the CP and the genome-linked protein are the solitary gene products in the virion [33].

Thus, in our study, we have taken the coat protein of YBMV for the structural and functional analysis of the virus CP. Since genomic structure differs significantly among families and the CPs display very low-level sequence similarity, it is fascinating to explore the virion architecture. Through integrative modeling, we explored the plausible organization of YBMV CP and its mode of single-stranded RNA (ssRNA) recognition followed by the all-atoms molecular dynamics simulation (MDS) for a 50 ns timeframe. We also identified a few conserved residues that aid in binding of RNA which suggests antiviral compounds could be targeted to these regions which may profound effect on the genome assembly and packaging of economically important viruses like YBMV. As a consequence of our research, we now have a better understanding of the structural basis of CP assembly in YBMV, which will help us in developing new antiviral drugs that target the conserved fold of RNA binding.

Materials and methods

Primary sequence analysis

The FASTA format amino acid sequence of YBMV CP was obtained from the NCBI protein database with accession number YP_004940328. The coat protein functional region/domain region was identified using the Conserved Domain Database (CDD) [34]. The phosphorylation site was identified using the NetPhos 2.0 server [35]. To gain broad chemistry of YBMV CP, a clear study on the primary sequence was carried out using the ProtParam tool [36] of Expasy Proteomic Server. The ProtParam provides information on different Physico-chemical properties of the coat protein, such as instability index, molecular weight, iso-electric point, grand average hydropathy (GRAVY), and aliphatic index.

Molecular evolutionary analysis of YBMV CP

The molecular evolutionary analysis of YBMV, a phylogenetic tree was built using the NJ technique in MEGA version 6 software [37]. A total of twenty-six homologous coat protein sequences of potyvirus were collected using the DELTA-BLASTP search against the non-redundant (nr) database to construct the evolutionary genetic tree. Percentage of trees replicated where the accompanied taxa grouped in the bootstrap test (containing 1000 replicates) were signified next to the branches. The position correction method is used for determining the protein evolutionary distances through gap extension penalty, gap separation distance, and gap opening penalty employing Blossum weight matrix. All locations encompassing gaps and missing data were removed throughout the study. Using Clustal W software, multiple sequence alignment was conducted on fourteen coat proteins of Potyviruses [38].

Secondary structural analysis

The PSIPRED server was used to identify the secondary structure of YBMV CP [39]. PSIPRED helps in sequence annotation by TM (transmembrane) domain mapping and secondary structure mapping [40]. This server is one of the specific secondary structure prediction methods that includes two feed-forward neural networks in which the analysis is based on the output obtained from Delta-Blastp.

Template identification

Suitable templates were detected for the comparative modeling of YBMV CP, the target sequences of YBMV CP were investigated for similar sequences using the Blast-P i.e., against the PDB database [41]. The matrix used for the template identification is the Blossum-62 matrix with a default inclusion threshold value (0.005) and E-value (10). The templates were chosen based on the sequence identity, less E-value, query coverage, and structural resolution. After the selection of an appropriate template, a target-template alignment was performed for the generation of protein structure.

Computational modeling

Arnold and co-authors have developed the SWISS-MODEL workspace with a range of tools that permits the user to modify and validate the various modeling steps manually. The SWISS-MODEL workstation includes application software required for 3D structure in an easy-to-use web-based modeling computer unit. A 3D model for the target protein is generated based on the sequence alignment among the target and template structures. For structure creation, SWISS-MODEL was used to model the 3D proteins [42].

Model validation

An essential web application such as PROCHECK has been deployed to explain the authenticity, stereochemical consistency, and durability of the projected 3D model [43]. PROCHECK was used for accessing the geometry of the model. Protein structure analysis i.e ProSA [44] was applied for calculating the energy potential of the predicted model, and the Verify3D [45] score used for the analysis to check the quality of the predicted model, It’s also used to check if the atomic model is compatible with its amino acid sequence. Mol Probity [46] is a web service for structure validation providing a broad-spectrum solidly based evaluation of model quality at both the local and global levels for both nucleic acids and proteins [46]. In comparison to other approaches that are optimized to find native structures, ERRAT [47] was performed for estimating the accuracy of the non-bonded atoms of the modeled YMBV. The validation results were interpreted, accompanied by re-validation to achieve an initial optimized model structure, and the structural ambiguities were resolved.

Identification of RNA binding pocket

To identify the plausible mode of RNA recognition, the homo-oligomeric assembly (24-mers) [48] using monomeric sub-units of modeled CP of YBMV was built. Based on the potyvirus virion structure (PDB: 5DOV) [49], the oligomeric structure of CP sub-units was modeled and transferred the coordinates of RNA to our modeled structure using PyMOL. The electrostatic surface potential map was constructed using the APBS plug-in of PyMOL. To identify, the conserved RNA binding site which was reported in earlier studies [48]. We have employed HADDOCK v2. 4 web server to predict the RNA binding affinity with both mutant and wildtype [50-52].

Molecular dynamics simulation

The stability and dynamic behavior of the modeled structure were investigated based upon the Desmond with 2005 force field i.e., (OPLS) Optimized Potentials for Liquid Simulations [53]. To examine the multiple conformations and interactions of the ligands within the binding pocket of YBMV CP, we used Desmond molecular dynamics simulations. TIP3P was used as a solvent in an orthorhombic box and the protein was neutralized by introducing counter ions and 0.15M of salt concentration to neutralize the protein [54]. The distance between the box wall and the protein was fixed at 10 Å microns to avoid steric interference with the protein periodic images. Energy minimization was performed using a hybrid approach of steepest descent and limited memory Broyden–Fletcher–Goldfarb–Shanno (LBFGS) algorithms with a maximum of 5000 steps until a gradient threshold was reached up to 25 kcal/mol [55]. The NVT (number of particles, Volume, and Temperature) ensemble conditions were kept the same for 1ns to get simulation data for post-simulation analysis [56]. Nose-Hoover thermostats were used to keep the temperature at 300 degrees Celsius throughout the simulation, and the Martyna-Tobias-Klein barostat method was employed to keep the pressure at a consistent level throughout. A multi-step RESPA integrator technique was utilized to investigate the equation of motion in dynamics [57]. The completed equilibrated system was used to run a 50 ns MD simulation, which was examined using Desmond’s event analysis module. The docking complexes were employed as starting structures for a 50 ns MD simulation, which was performed on them. Structures were imported into Desmond’s set-up wizard and soaked with TIP3P inside an orthorhombic box, like a box with a top and bottom. All other procedures were carried out in the same manner as described in the free form of the YBMV CP protein simulation.

Results

Sequence analysis of YBMV CP

Yam bean mosaic virus (YBMV) coat protein (CP) sequence was retrieved from the NCBI protein database (YP_004940328) consisting of 275aa. The domain prediction using SMART, Pfam, and CDD (conserved domain database) revealed a common conserved region of the coat protein sequence of the YBMV i.e. (41-270aa) 230aa. This conserved region is utilized further for the structural analysis. The primary sequence analysis of YBMV CP (230aa long) illustrated that the protein is acidic (an isoelectric point is 6.26) in nature and has a molecular mass of 26.41kDa. The aliphatic index is 75.13 which indicate the protein’s stability over a wide range of temperature. It is widely recognized that a protein with an instability index less than 40 is considered to be stable, while if the value ranges above 40 it shows the protein to be unstable [58]. The coat protein (CP) of YBMV is stable as its instability index is 37.02 (i.e., <40). The protein contains 31 negatively charged and 29 positively charged amino acids. YBMV CP has a Grand Average Hydropathicity GRAVY value of -0.576, indicating that it may have better contact with water. The phosphorylation sites of YBMV CP protein were determined using NetPhos 2.0 [35] which indicates 5 serine, 5 threonine, and 2 tyrosine sites for phosphorylation.

Secondary structure analysis and 3D prediction

The secondary sequence analysis reveals that, sequence annotation is influenced by expected features like TM and secondary structure mappings. PSIPRED performs the secondary structure analysis where the coils/turns get dominated over strands and helices along with the secondary structure elements. The secondary structure map discovers the amino acid residues which have the propensity to be a beta-sheet or an alpha-helix are present in the proteins derived from the YBMV CP sequence. In addition, sequence characteristics such as the DomPred boundary, disordered area, disordered protein binding sites, and DomSSEA boundary are discovered and annotated in the sequence ( The protein sequence of YBMV CP shows the presence of the transmembrane helix region starting from 76aa up to 91aa. It also depicts the disordered protein binding area, which spans 215 to 233 amino acids. Sequence features like cytoplasmic region, pore-lining helix and extracellular region are annotated and predicted. The contact maps of YBMV CP revealed the amino acid residue-residue interactions ().

Phylogenetic analysis

To understand the molecular evolution of YBMV from Pachyrhizus erosus, phylogenetic analysis was performed on 26 different coat proteins of Potyviruses belonging to the family Potyviridae (see The Neighbor-Joining method with 1000 iterations was used to create the 2D phylogenetic tree and branch length 4.229 has been displayed using MEGA version 6 (. The Poisson correction technique was used to compute the evolutionary distances. The phylogenetic cladogram represents altogether five clusters and three major clusters formation. On the other hand, it was found that the CP sequence of YBMV shows closely related to CP of Soybean mosaic virus, Bean common mosaic virus and Watermelon mosaic virus. Clusters 3, 4 and 5 show highly dissimilar clades i.e., less sequence similarity to YBMV coat protein. Multiple sequence alignment was performed using Clustal W with the coat protein sequences of Yam bean mosaic virus, Dasheen mosaic virus, Leek yellow stripe potyvirus, Chilli veinal mottle virus, Watermelon mosaic virus, Beet mosaic virus, Onion yellow dwarf virus, Bean common mosaic virus, Cocksfoot streak virus, Bean yellow mosaic virus, Cowpea aphid-borne mosaic virus, Johnson grass mosaic virus, Soybean Mosaic virus, Lily mottle virus, Maize dwarf mosaic virus, Papaya ringspot virus, Peanut mottle virus, Yam mosaic virus, Japanese yam mosaic virus, Zucchini yellow mosaic virus, Turnip mosaic virus, Wild potato mosaic virus, Sugarcane mosaic virus, Sweet potato feathery mottle virus, Sorghum mosaic virus. Multiple sequence alignment of YMBV CP (coat protein) sequence with other CP viruses from the potyvirus family (.

The evolutionary relationship of taxa of YBMV CP and other closely related organisms using the NJ method.

The bootstrap test (1000 repetitions) showed the percentage of replicate trees where the related taxa were grouped. The analysis was conducted using MEGA version 6.0 software.

3D modeling

The search for the templates for YBMV CP in PDB using Delta Blastp explained the crystal structure of Chain A, Watermelon Mosaic Virus (WMV) coat protein (PDB ID: 5ODV), Chain: A [48] shared the highest homology with an identity with coat protein (Bean common mosaic virus) of 80.44% and Query coverage 97%; Coat protein (Peanut stripe virus) identity 79.57 and Query coverage of 97%. The target-template alignment was conducted using the ESPript 3.0 programme as the comparative modelling hinges on a sequence alignment between the template and the target sequence with an experimentally determined structure (). Based on the target-template alignment, the protein model was constructed by the SWISS-MODEL web server.

Structure validation

The protein structure of the YBMV coat protein comprises two β-strands (one parallel and one anti-parallel) and eight α-helices. The outer surface of the protein is surrounded by the α-helices and the inner core of the model is built of β-sheets (). The dihedral angles (Φ/Ψ) of the YBMV coat protein’s accuracy were measured by using the Ramachandran plot in Procheck program (). Secondary structure analysis was performed using the PROCHECK analysis tool thus representing the alpha helices and beta sheets in a distinct manner. The 3D model represented a good percentage of residues in favored regions (86.2%), 13.5% residues in additional allowed regions, 0% residues in disallowed regions and 0.5% residues in allowed regions. ERRAT is used in the statistical calculation of the interactions between different atoms types, it also reveals a good validation score is 90%. ERRAT suggests the reliability of the initial model. The ProSA software aims to obtain the proposed model’s energy profile as well as the z-score value, which further helps to measure the energy of residue relationship. ProSA evaluated a z-score rating value i.e., as depicted in . Protein topology map, constructed using Procheck, with beta-strands as a pink-colored arrow and the alpha-helices as red cylinders (. The details of the protein structure validation are summarized in .

(A) The three-dimensional structure of YBMV CP N-terminal region (red); C-terminal region (blue). (B) The protein is visualized as a solid ribbon representing β‐sheets, α‐helices, and coils.

(A) Ramachandran plot of the YBMV CP model. The plot was created using the PROCHECK program. (B) Protein Structure Analysis (ProSA) of model YBMV CP. The overall quality of the YBMV CP model shows the z-score value of -3.75 (Native conformation to its template).

Oligomeric assembly of the conserved fold of YBMV coat proteins

It has been observed that 24 CPs monomeric sub-units form a conserved fold. As a result, we produced its oligomeric form based on the template in our research. The method [53] is based on supervised machine learning and Supports Vector Machines, which integrate interface conservation with other template characteristics structural clustering to produce a quaternary structure quality evaluation. The QSQE score is a number between 0 and 1 that indicates the predicted accuracy of interchain interactions for a model generated with a certain alignment and template. The greater numbers imply more reliance. This is added to the GMQE score, which measures the correctness of the resultant model’s tertiary structure.

RNA binding sites

Interactions between the CP and the viral RNA, in particular, must be tightly controlled for the RNA to be correctly assigned to each of its functions throughout the infection process. In the case of YBMV CP, each subunit covers nucleotides of ssRNA (). The presence of a conserved binding site is suggested by analyzing the seven amino acid residues present in the atomic structure of the YBMV CP. The key residues interacting with wild type RNA binding sites are LYS 188, VAL 172, GLU 171, ASN 197, ARG 124 and THR 122 respectively (). The key residues interacting with mutant type RNA binding sites are LYS 188, SER 92, THR 91, PRO 93, ASP 168, TYR 170, ALA 142, LEU 123 and THR 122 respectively (). The intermolecular contacts of YBMV CP with RNA were also determined using SWISS-MODEL shown in .

Oligomeric assembly of 24 identical subunits of CP with RNA (A) top, (B) front and (C) surface representation).

Interaction analysis of YBMV CP with RNA binding reorganization.

(A) Wild type; (B) R124A mutant.

A total of 132 structures in 11 cluster(s), which represents 66% of the water-refined models HADDOCK generated. The wild type HADDOCK score (-68.7), Van der Waals energy (-38.6), Electrostatic energy (-218.7) respectively. The total of 160 structures in 8 cluster(s), which represents 80% of the water-refined models HADDOCK generated. The mutant type HADDOCK score (-61.9), Van der Waals energy (-40.6), Electrostatic energy (-166.2) respectively.

Molecular dynamics simulation of YBMV CP

Several dynamic stability parameters, including the RMSD, Cα‐RMSF, Rg of intermolecular H-bonds, were examined with simulation time after the protein and complex of YBMV CP were obtained. RMSD evaluated the initial structure by MD simulation for 50 ns to investigate the effective confirmation of hits, protein stability, and RNA-binding recognition. By analyzing the deviation caused during the simulation, the Root-mean-square deviation (RMSD) was able to estimate the protein’s stability in comparison to its initial structure. The RMSD value shows that, the RNA complex has a more stable shape. The average RMSD of the complex and 3D model of YBMV CP was computed 50 ns after the start of the simulation. RMSD for complex 30 ns to 50 ns stable within 4Å while apo protein deviated throughout the simulation from 6–7.5 (. YBMV CP was produced in both the apo and holo forms, and the C-alpha atom’s RMSF profile for each residue was computed to determine the residue-wise mobility during the simulation. The YBMV CP-RNA system showed more flexibility as compared to the YBMV CP. The complex’s C-alpha Root mean square fluctuations (RMSFs) demonstrated that high fluctuation and confirmed the RMSD results. The mean variation plot was used to assess the compounds average RMSF value. The RMSF for the protein complex C-alpha displays less variation than the RMSF for the apo protein (). From the examination of the radius of gyration (Rg), it is clear that the compactness of the complex protein was stabilized between 17.0–17.5 Å, but the compactness of the apo protein was disturbed throughout the simulation. Protein and RNA complexes’ compactness and dimensions are determined by it, and this may explain why they interact well. Protein and RNA complexes compactness and dimensions are determined by it, and this may explain why they interact well (). H-bonds are the most essential factors for biomolecular complexes’ stability. It was determined that the number of discrete H-bonds formed between certain amino acid residues of the YBMV core protein and RNA atoms was computed using the Desmond with Optimized Potentials for Liquid Simulations (OPLS) 2005 force field. We computed the intermolecular H-bonds generated between YBMV CP and the RNA-Complex as a function of the simulation timeframe. The H-bond study revealed that RNA created 8–12 hydrogen bonds with protein during the replication process (). Overall, taking into consideration of dynamics stability, H-bond analyses and the pre and post-MD RNA binding views, it may be concluded that was more stable.

Conformational stability of YBMV CP of apo and complex through MD simulation.

(A) Backbone RMSD (B) C-alpha RMSF YBMV CP (C) Radiation of gyration for YBMV CP; (D) Hydrogen bond analysis over 50 ns period concerning the index of the residue.

Wild type

The ARG side chain has four structure-based donors that can all hydrogen-bond to a single H-bond acceptor.

Asp 168 showed more than 95% direct interaction and formed water mediated interaction more than 35% with the RNA during simulation. Arg124 established 3 direct interactions with the RNA and stabilized more than 60% of the molecular dynamics simulation (). represent the different bond interaction of the amino acid resides with RNA. Arg124 interacted with RNA by forming mostly H-bond and water bridge. AGR 155 communicated with RNA through H-bond, salt bridge and water bridge. The bottom panel shows which residues interact with the ligand in each trajectory frame. Some residues make more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot ().

The H-bond occupancy of wild type YBMV CP with RNA throughout the trajectory.

(A) 2D interaction of RNA with YBMV CP during simulation (B) the amino acid residues involved in the interactions (C) the overall interaction of the protein with RNA.

Mutant type MD simulation

We have used different stability predictors to assess the effect of mutation of the key residues. The RMSD value shows that the R124A and complex has a more stable during 50 ns MD simulation. RMSD for complex 15 ns to 45 ns stable within 4Å while apo protein deviated throughout the simulation (. The complex’s C-alpha Root mean square fluctuations (RMSFs) demonstrated that high fluctuation and confirmed the RMSD results. The RMSF for the R124A and complex C-alpha displays less variation than the RMSF for the apo protein (). The examination of the radius of gyration (Rg), it is clear that the compactness of the complex protein was stabilized between 17.0–15.5 Å, but the compactness of the R124A protein was disturbed throughout the simulation (). H-bonds are the most essential factors for biomolecular complexes’ stability. It was determined that the number of discrete H-bonds formed between certain amino acid residues of the R124A and R124A Complex was computed using the Desmond with Optimized Potentials for Liquid Simulations (OPLS) 2005 force field. We computed the intermolecular H-bonds generated between R124A and R124A Complex as a function of the simulation timeframe. The H-bond study revealed that created 2–14 hydrogen bonds with protein during the replication process (). Overall, taking into consideration of dynamics stability, H-bond analyses and the pre and post-MD RNA binding views, it may be concluded that was more stable.

Conformational stability of YBMV CP of R124A_Apo and R124_Complex through MD simulation.

(A) Backbone RMSD (B) C-alpha RMSF YBMV CP (C) Radiation of gyration for YBMV CP; (D) comparison of Hydrogen bond analysis between wild type and R124A mutant over 50 ns period concerning the index of the residue.

Mutant type

The disclosed that Ala124, Leu123, Pro121, Tyr170, Lys180 and Thr176 established direct interaction with the RNA and stabilized more than 30% throughout simulation. Additionally, it has been noticed that ARG155 communicated with RNA through H2O molecule. Furthermore, distribution of interaction of each amino acid residues has been displaced in . A timeline representation of the interactions and contacts H-bonds, Hydrophobic, Ionic, Water bridges. The bottom panel shows which residues interact with the RNA in each trajectory frame. Some residues make more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot ().

The H-bond occupancy of mutant type YBMV CP with RNA throughout the trajectory.

(A) 2D interaction of RNA with YBMV CP during simulation (B) the amino acid residues involved in the interactions (C) the overall interaction of the protein with RNA.

Discussion

Among legumes, yam beans (Pachyrhizus erosus) have the greatest adaptability and production potential [10]. Potyvirus is considered to be the largest and economically most important plant virus. The potyvirus consists of 28 plant virus families and groups. In 1974 total of 1112 species of 369 genera in 53 plant families has been reported [30]. One-quarter of known plant RNA viruses belong to the potyvirus group family potyviridae [59, 60]. Presently there are 2,026 no. of plant species and 566 no. of genera along with 81 botanical families that are globally distributed, which are subjected to potyviruses [61]. More than 200 species of aphids are responsible for the transmission of the potyvirus occurring in a non-persistent way [59, 62]. As a result, potyviruses have a unique capacity to adapt to novel habitats, hosts, and vectors, according to Nigam and co-authors. The investigation of polyprotein-wide and genome-wide variation indicated that the potyviral genome possesses hypervariable locations that preferentially collect nucleotide substitutions and sites under positive selection, encouraging protein disorder and perhaps determining host adaptability [63]. The potyviruses are found in numerous pathogens or strains which vary in biological properties i.e pathogenicity [64]. According to Fuentes et al., (2012), a new virus known as Yam Bean Mosaic Virus (YBMV) was discovered and named, and it belongs to the potyviridae family. The virus was first reported by [19], and by default, the name was given as BCMV-IYbn. Latter in 2012 after further study on the virus Fuentes discovered the virus in the yam bean crop and named it Yam Bean Mosaic Virus (YBMV). There is no clear identity, of the YBMV CP core amino acid sequence with the corresponding sequences of members of the genus Potyvirus. It is necessary to achieve a better knowledge of the coat protein structure of YBMV which may help to understand the virus’s multiple functions. The N-terminal portions of potyvirus CP sequences are the most diverse, whereas the C-terminal half is very conserved [62]. Thus, the capsid protein and 3’ NCR sequences are the most essential parts of the potyviral genome for taxonomic study, and the 3’-terminal area may be utilized to identify virus strains from other potyviruses, according to all molecular data [62, 65].

According to Shukla and Ward (1989), the nucleotide sequence of the CP gene among strains of a certain potyvirus species was more than 90% similar [31], whereas the degree of similarity in the nucleotide sequences of the 3’ UTR across strains of specific potyvirus species ranged from 83–99 percent, whereas different virus species had identities ranging from 39–53 percent, according to Frenkel et al (1989) [66]. Adams et al 2005 looked at several potyvirus sequences and determined that the greatest possible CP nucleotide sequence identity in the same potyvirus species was 76–77 percent, while the highest possible CP amino acid sequence identity was 82 percent [67].

The virus is described as a new, positive-sense, single-stranded RNA virus enveloped in a novel way [19]. Instead of the full-length sequence, only the domain region was taken for the structural analysis for accurate optimization. The viral sequence is depicted as a novel enveloped, negative-sense as well as single-stranded RNA virus whose strain belongs to the country named Peru. The virus comes under the potyvirus group, a family named potyviridae. The overall small RNA size distribution revealed a significant preponderance of 21-nt RNAs, which is consistent with prior severe potyviral infections [20]. A phylogenetic analysis was carried out representing five clusters from which two are considered to be major clusters. From our study, it was found that the YBMV CP shows high similarity with Bean Common Mosaic Virus (BCMV) and then secondly with Soybean Mosaic Virus and then with Watermelon Mosaic Virus, the remaining cluster illustrates highly dissimilar clades. Currently, the isolate taken for the analysis is from Peru [22] and the respective amino acid sequence is available in NCBI. The primary structure analysis, as well as the sequence annotation, is essential for converting the sequence data to functional genes which is essential to understanding the functional and biological mechanisms [68]. In addition to direct functional analysis, the precise position of the transmembrane helices (TM) is important for functional annotation [69]. The YBMV contains the TM region starting from 76aa up to 91aa. For understanding, the functional and the biological mechanisms sequence annotation and physiochemical property analysis are essential, which are obtained after converting the sequence data into functional genes [70]. Preliminary attempts were made to obtain structural information on the coat protein of YBMV by using in-silico approaches yielding certain results.

It is important to know how proteins work in three dimensions to answer many biological questions. However, the number of genes and genomes that have been sequenced is quickly outpacing the number of experimentally determined structures. In this approach, comparative modeling continues to be a highly effective strategy of choosing. It contributes to the closure of the gap between what we know about the sequence of a protein and how it appears by creating precise and trustworthy protein models [71].

To solve many biological problems there is a need to have good knowledge regarding the three-dimensional protein structure of the virus (YBMV). Nowadays, comparative modeling is performed as it is an increasingly important method to obtain a suitable 3D protein structure [70, 72–75]. Comparative modeling’s main benefit is that it aids in filling in gaps between available sequence and structural data by generating a reliable and accurate protein model [71]. The approach and parameters of MD simulation were adapted from our early studies to better comprehend the molecular function of unknown/hypothetical molecules [76-81].

In our work, 3D modeling is performed following the template search for YBMV coat protein (PDB ID: 5ODV, Chain: A) [48], resulting in appropriate 3D models for a wide range of applications. However, the model, structural stability, and protein-ligand interaction studies also provide prior knowledge for treatments creation soon. Molecular dynamics simulation provides a very realistic, safe, and straightforward atomic level technique for understanding protein folding and dynamics over a certain timescale [82].

Using a bioinformatics approach, several computational tools are provided to examine various dynamical behaviours of the protein structure. In this work, we examined our protein and the complex of YBMV CP utilizing several characteristics such as RMSD, CRMSF, and Rg of intermolecular H-bonds. The original structure was analyzed for RMSD using MD simulation for 50ns to study the effective confirmation of hits, protein stability, and RNA-binding recognition. The average root mean square deviation (RMSD) of the complex and three-dimensional model of YBMV CP was calculated 50ns after the simulation. The apo protein’s RMSD fluctuated from 6–7.5 throughout the experiment, while the RMSD for the complex remained steady at 4. (. Less variation is seen in the RMSF for C-α, a complex of proteins, than in the RMSF for apo C-α. (). The radius of gyration (Rg) shows that the compactness of the complex protein was stable between 17.0–17.5, but the compactness of the apo protein was not stable at all (). H-bonds are the most important variables in determining the stability of biomolecular complexes. A force field based on the Desmond with Optimized Potentials for Liquid Simulations (OPLS) 2005 force field was used to calculate the number of discrete H-bonds generated between specific amino acid residues of the YBMV core protein and RNA atoms. According to this research, we computed the intermolecular H-bonds produced between YBMV-CP and the complex of RNA molecules as a function of the simulation period. It was discovered through the H-bond study that RNA formed 8–12 hydrogen bonds with protein during the replication process ().

The emergence of this virus disease might pose a significant threat to India’s newly launched and promising yam bean Mosaic Virus output. Other significant factors to assess the threat of this virus to global yam bean output include the virus’s worldwide prevalence, variability, susceptibility, and influence on the yield of different yam bean species. Our results will also help in the development of novel antiviral medicines that target the RNA binding pocket, which might disrupt the genome packaging and assembly of economically significant plant viruses like YBMV [48]. Overall, we anticipate that our unique insights into RNA interaction mechanism in Yam bean Mosaic Virus (coat protein), as well as the new study on virus particles, will lead to control of potyviral infection in the future.

Conclusions

Understanding the significance of a viral protein in the infection process involves a detailed three-dimensional structure of the protein. Our study substantiates from the molecular evolutionary analysis that the YBMV CP of yam bean is highly similar to the coat protein of Bean common mosaic virus, Soybean mosaic virus, Watermelon mosaic virus and potyviridae family. We systematically searched the desired YBMV CP sequence for the in-silico analysis and homology-driven structure prediction was generated and characterized in yam bean. The emergence of this new viral illness might pose a significant danger to the yam bean production that has only lately begun and appears to be promising. The entire genome of a novel potyvirus could be reconstructed from the siRNA sequences, and it was distinct enough from other sequences to be classified as a new species. Future research should focus on developing precise detection methods and determining the degree of seed transfer so that suitable phytosanitary measures may be developed and implemented to prevent the disease from spreading further. Other critical characteristics needed to assess the threat of this virus to global yam bean output include the virus’s worldwide prevalence, variability, susceptibility, and influence on the yield of diverse yam bean species.

The secondary structure map.

The figure represents the secondary structure map where query sequence and coloured residues are presented as per the annotations made by each analysis methods. Map shows helical residues that are coloured in pink, b-strand residues coloured in yellow, putative domain boundaries are indicated in blue, and green bordered box represents disordered protein binding.

(TIF)

Click here for additional data file.

The contact map.

The figure shows the contact map of YBMV protein model. The map shows a matrix with contacts with the color scale which represents the relationship between the amino acids in the particular map.

(TIF)

Click here for additional data file.

Multiple sequence alignment.

Multiple sequence alignment of YMBV_CP (coat protein) sequence with other CP viruses from the potyvirus family. The image is generated in Multalin software.

(TIF)

Click here for additional data file.

Protein topology map.

Protein topology map, constructed using Procheck, with beta-strands as a pink-coloured arrow and the alpha-helices as red cylinders.

(TIF)

Click here for additional data file.

Identification of protein domain.

The full-length sequence acc. no. of the coat protein of potyvirus and identification of protein domain using the conserved domain search.

(DOC)

Click here for additional data file.

16 Mar 2022

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Title; Structural insights into the RNA interaction with Yam bean Mosaic Virus (coat protein) from Pachyrhizus erosus using bioinformatics approach.

Comments;

1. Authors have not provided the name of the template used for model generation in the abstract.

2. Authors have suggested to rewrite the results and conclusion part of the abstract based on findings. In addition, the authors have advised adding a brief description of methodology in the abstract.

3. Objective of the study should be clear at the end of the introduction section.

4. Authors have been advised to correct the spelling mistakes throughout the manuscript. For example “formatted”.

5. In the Introduction section the author should refer to the research paper and comment on recent in-silico techniques. It will be good information for the readers. I would like to recommend several papers, among many others, providing further explanation on this topic:PMID: 34717229 PMID: 33465692 PMID: 33749525 PMID: 29380245 PMID: 31933177 PMID: 32353478 PMID: 33993976

6. Provide citations for all the PDB IDs mentioned in the manuscript.

7. Authors should include studies with RNA on the predicted binding site. This could be done by docking and MD simulations.

8. MD simulations should be carried out to identify key structural changes and residues involved in protein-RNA interactions.

9. Clearly mention the aim and objective of the study in the last paragraph of the introduction section.

10. Including limitations of the study in the discussion and conclusions section.

11. Authors have not provided references regarding servers used in the studies.

12. Figure 6 is not visible authors have advised redrawing the figure.

13. Authors have advised to include more rigorous analysis and validate the findings by experimental/computational analyses.

18. Authors have not compared their findings with already present literature based on similar studies.

19. Overall, the manuscript is written in a very confusing and poor way. The writing needs substantial improvement. Need to edit the English language through a native speaker or by other means.

In my view, the results obtained in this study are worthy for publication. The manuscript needs major essential revision before publication. I would like to overview the revised version of the manuscript before it accept for publication.

Reviewer #2: In this manuscript titled “Structural insights into the RNA interaction with Yam bean Mosaic Virus (coat protein) from Pachyrhizus erosus using bioinformatics approach” authors have modeled the 3D structure of CP and merely predicted the amino acid that is involved in interaction with RNA. There is no experimental evidence for their finding. I recommend the authors plan a wet lab experiment to demonstrate the role of those amino acids in RNA binding. For instance, make mutation of those amino acid residues by SDM and test the ability of mutant proteins to bind RNA by EMSA, fluorescent spectroscopy, etc. The manuscript is not written well.

Specific comments:

Page 3, Line 15: “Chenopodium amanticolor, Nicotiana megalosiphon and Nicotiana benthamiana”. Scientific name should be italicized.

Page 3, Line 80: “Sorensen” since there are more than one author in this study, you should mention as Sorensen et al.,

Page 3, Line 86: Similar to previous comment. Change “Damayanti” to ““Damayanti et al”

Page 4, Line 88: “Afterward in……”. Not scientific. Rephrase this sentence.

**********

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Reviewer #1: No

Reviewer #2: Yes: PRABU GNANASEKARAN

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20 Apr 2022

Manuscript Number: PONE-D-22-04164

Point-to-point response to the Reviewers' comments

We thank the editor for the positive evaluation of our paper, and the two reviewers for recognizing the merit of our work and for providing very valuable suggestions. As detailed below, we have addressed the Reviewers’ comments carefully and revised the manuscript accordingly. To facilitate the reviewing process, we have copied the Reviewers’ original comments, which are shown in black (italic font) and our responses are then shown in blue.

Reviewer #1

Comments 1

Authors have not provided the name of the template used for model generation in the abstract.

Authors Response

The template used for the model generation is 5ODV. We have added the template name in the abstract section of the Ms.

Comments 2

Authors have suggested to rewrite the results and conclusion part of the abstract based on findings. In addition, the authors have advised adding a brief description of methodology in the abstract.

Authors Response

As per the suggestion, the results, conclusion and methodology part has been revised accordingly in the abstract section of the Ms.

Comments 3

Objective of the study should be clear at the end of the introduction section.

Authors Response

Thank you for your response. We have modified the introduction section as desired by you.

Comments 4

Authors have been advised to correct the spelling mistakes throughout the manuscript. For example, “formatted”.

Authors Response

We have corrected our spelling mistakes throughout the manuscript.

Comments 5

In the Introduction section the author should refer to the research paper and comment on recent in-silico techniques. It will be good information for the readers. I would like to recommend several papers, among many others, providing further explanation on this topic: PMID: 34717229 PMID: 33465692 PMID: 33749525 PMID: 29380245 PMID: 31933177 PMID: 32353478 PMID: 33993976

Authors Response

Thank you for your valuable comments. These publications are highly useful regarding in silico techniques and we have also cited the same in the Ms accordingly.

Comments 6

Provide citations for all the PDB IDs mentioned in the manuscript.

Authors Response

We have cited all PDB IDs in the Manuscript.

Comments 7

Authors should include studies with RNA on the predicted binding site. This could be done by docking and MD simulations.

Authors Response

Thank you for your suggestion. We have performed an MD simulation of 50ns using Desmond with Optimized Potentials for Liquid Simulations (OPLS) 2005 force field.

Comments 8

MD simulations should be carried out to identify key structural changes and residues involved in protein-RNA interactions.

Authors Response

As suggested, we performed an MD simulation to analyze the key structural changes and residues involved in protein-RNA interaction in the Ms.

Comments 9

Clearly mention the aim and objective of the study in the last paragraph of the introduction section.

Authors Response

The aim and objective of the study have been rectified and explained properly in the introduction section of the Ms.

Comments 10

Including limitations of the study in the discussion and conclusions section.

Authors Response

We have modified the discussion and conclusion section as per your suggestion.

Comments 11

Authors have not provided references regarding servers used in the studies.

Authors Response

Thank you for your recommendation. We have cited some references in the Manuscript.

Comments 12

Figure 6 is not visible authors have advised redrawing the figure.

Authors Response

We have revised figure 6 as per your suggestion.

Comments 13

Authors have advised to include more rigorous analysis and validate the findings by experimental/computational analyses.

Authors Response

We have performed a molecular dynamics simulation to validate more findings in computational bioinformatics techniques.

Comments 14

Authors have not compared their findings with already present literature based on similar studies.

Authors Response

We have revised the Manuscript with recent literature studies.

Comments 15

Overall, the manuscript is written in a very confusing and poor way. The writing needs substantial improvement. Need to edit the English language through a native speaker or by other means.

Authors Response

We apologize for the inconvenience. We have revised the manuscript accordingly. We have used Grammarly software and corrected the spelling and grammatical errors present in the manuscript and rewritten the portion that was confusing for the reviewer.

Reviewer #2

Comments 1

There is no experimental evidence for their finding. I recommend the authors plan a wet lab experiment to demonstrate the role of those amino acids in RNA binding. For instance, make mutation of those amino acid residues by SDM and test the ability of mutant proteins to bind RNA by EMSA, fluorescent spectroscopy, etc.

Authors Response

Thank you very much for your recommendation. We agree with the reviewer on the above remarks that, the findings from the computational study should be validated through an experimental approach. We are a small group of researchers working in computational biology without the support of a wet-lab experimental setup, therefore, we apologize that the suggested experiments by the esteemed reviewer could not be performed at present MS. However, we strongly believe that our computational findings will be helpful for the researchers working in this area to plan and undertake such studies involving site-directed mutagenesis, EMSA and fluorescent spectroscopy studies which will unravel the binding of RNA to the protein. To additionally, support our study, we have performed all-atoms MD simulation for 50 ns to analyze the structural changes and residues involved in protein-RNA interaction and stability of the complex through RMSD, RMSF, Rg and H-bond analysis (Please see in Fig. 7 of the Ms).

Comment 2

The manuscript is not written well.

Authors Response

Thank you for your suggestion. We have revised the manuscript rigorously and rectified the grammatical mistakes throughout the manuscript.

Specific comments:

Comments 3

Page 3, Line 15: “Chenopodium amanticolor, Nicotiana megalosiphon and Nicotiana benthamiana”. Scientific name should be italicized.

Authors Response

We have corrected the scientific name in italics.

Comments 4

Page 3, Line 80: “Sorensen” since there are more than one author in this study, you should mention as Sorensen et al.,

Authors Response

We have revised the Sorensen as Sorensen et al., in the Ms.

Comments 5

Page 3, Line 86: Similar to previous comment. Change “Damayanti” to ““Damayanti et al”

Authors Response

We have corrected the “Damayanti” to “Damayanti et al” in the introduction section.

Comments 6

Page 4, Line 88: “Afterward in……”. Not scientific. Rephrase this sentence.

Authors Response

As per the reviewer's suggestion, we have rewritten the above line.

Submitted filename: Response to Reviewer.doc

Click here for additional data file.

29 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Rajarshi Gaur

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have responded to all the concerns meticulously and improved the manuscript accordingly. The revised draft is improved significantly. I do not have further comments.

Reviewer #2: Authors have addressed the comments partially. Still there is no strong evidence for their conclusion. I recommend authors to perform atleast few more computational analyses to support their conclusion.

Specific comments:

Still, there is no wet lab experimental evidence for their finding. But authors have performed MD simulation to analyze the structural changes and residues involved in protein-RNA interaction. However, I recommend the authors to add atleast few more computation analysis, such as,

Generate simulation of mutant protein structure

Determining the wildtype and mutant protein affinity for RNA by Haddock score calculation.

Identify the pocket that binds to RNA, and the amino acid residues involved in the interaction in both wild-type and mutant protein.

To perform these experiments, I suggest authors to cite and follow the following publications.

Gnanasekaran, P., Gupta, N., Ponnusamy, K., and Chakraborty, S. (2021). Betasatellite encoded βC1 protein exhibits novel ATP hydrolysis activity that influences its DNA-binding activity and viral pathogenesis. Journal of Virology, 95 (20): e00475-21.

Gnanasekaran, P., Ponnusamy, K., and Chakraborty, S. (2019) A geminivirus betasatellite encoded βC1 protein interacts with PsbP and subverts PsbP-mediated antiviral defense in plants. Molecular Plant Pathology, 20(7): 943-960.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: PRABU GNANASEKARAN

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

4 Jun 2022

Manuscript Number: PONE-D-22-04164R2

Point-to-point response to the Reviewers' comments

We thank the editor for the positive evaluation of our paper, and the two reviewers for recognizing the merit of our work and for providing very valuable suggestions. As detailed below, we have addressed the Reviewers’ comments carefully and revised the manuscript accordingly. To facilitate the reviewing process, we have copied the Reviewers’ original comments, which are shown in black (italic font) and our responses are then shown in blue.

Reviewer #1

Comments

The authors have responded to all the concerns meticulously and improved the manuscript accordingly.The revised draft is improved significantly. I do not have further comments.

Authors Response

Thank you for your possetive response.

Reviewer #2

Comments 1

Generate simulation of mutant protein structure

Authors Response

Thank you very much for your recommendation. As per your suggestion, we have generated the mutant structure of R124A which plays important role in the interaction of YBMV_CP with RNA and maintained stable connection throughout the simulation. All the simulation analysis have been included in the MS.

Comment 2

Determining the wildtype and mutant protein affinity for RNA by Haddock score calculation.

Authors Response

Thank you for your suggestion. We have revised the manuscript accordingly. The HADDOCK score for the wild type and mutant type have been presented in the manuscript.

Comments 3

Identify the pocket that binds to RNA, and the amino acid residues involved in the interaction in both wild-type and mutant protein.

Authors Response

We have identified the pocket that binds to RNA, and the amino acid residues involved in the interaction in both wild-type and mutant type.

Comments 4

To perform these experiments, I suggest authors to cite and follow the following publications.

Gnanasekaran, P., Gupta, N., Ponnusamy, K., and Chakraborty, S. (2021). Betasatellite encoded βC1 protein exhibitsnovel ATP hydrolysis activity that influences its DNA-binding activity and viral pathogenesis. Journal of Virology, 95(20): e00475-21.

Gnanasekaran, P., Ponnusamy, K., and Chakraborty, S. (2019) A geminivirus betasatellite encoded βC1 proteininteracts with PsbP and subverts PsbP-mediated antiviral defense in plants. Molecular Plant Pathology, 20(7): 943-960.

Authors Response

Thank you for your suggestion. We have cited the two references in the Manuscript.

Submitted filename: Response to Reviewer.doc

Click here for additional data file.

12 Jun 2022

Structural insights into the RNA interaction with Yam bean Mosaic Virus (coat protein) from Pachyrhizus erosus using bioinformatics approach

PONE-D-22-04164R2

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11 Jul 2022

PONE-D-22-04164R2

Structural insights into the RNA interaction with Yam bean Mosaic Virus (coat protein) from Pachyrhizus erosus using bioinformatics approach

Dear Dr. Pati:

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Table 1

Model validation scores of yam bean mosaic virus coat protein (YBMV CP).

Model Validation Tools	Model validation reports	Scores
PROCHECK	Most favored region (%)	86.2%
	Additional allowed region (%)	13.3%
	Generously allowed region (%)	0.5%
	Disallowed region (%)	0.0%
	Overall G-factor	0.18
Verify3D	Averaged 3D-1D Score > 0.2	61.35%
ERRAT	Overall quality (%)	90.00
ProSA	Z-score	-3.75
ProQ	Predicted LG score	8.547
	Predicted Max Sub	-0.257
Molprobity	Ramachandran outliners (%)	0.00%
	Poor rotamers (%)	0.00%
	Bad bonds (%)	0.29%
	Bad angles (%)	1.34%

Table 2

Intermolecular contacts YBMV CP with RNA.

Pairs	Distance	Type	Category
A:ARG124:NH2—B:U3:OP2	2.17	Hydrogen Bond;Electrostatic	Salt Bridge
A:ARG124:NH2—B:U4:OP2	3.57	Hydrogen Bond;Electrostatic	Salt Bridge
A:ARG155:NH2—B:U5:OP1	3.04	Hydrogen Bond;Electrostatic	Salt Bridge
A:ARG124:NH1—B:U2:OP1	5.55	Electrostatic	Attractive Charge
A:ARG124:NH1—B:U3:OP1	5.59	Electrostatic	Attractive Charge
A:SER92:OG—B:U4:OP1	2.04	Hydrogen Bond	Conventional Hydrogen Bond
A:THR122:OG1—B:U3:OP1	3.08	Hydrogen Bond	Conventional Hydrogen Bond
A:TYR151:OH—B:U4:O2	2.65	Hydrogen Bond	Conventional Hydrogen Bond
B:U5:H3—A:ASP94:O	2.43	Hydrogen Bond	Conventional Hydrogen Bond
B:U3—A:LEU192	5.26	Hydrophobic	Pi-Alkyl
B:U4—A:LYS188	5.23	Hydrophobic	Pi-Alkyl