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Evolutionary and Molecular Aspects of Indian Tomato Leaf Curl Virus Coat Protein

Evolutionary and Molecular Aspects of Indian Tomato Leaf Curl Virus Coat Protein Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2012, Article ID 417935, 12 pages doi:10.1155/2012/417935 Research Article Evolutionary and Molecular Aspects of Indian Tomato Leaf Curl Virus Coat Protein 1 1 1 Sivakumar Prasanth Kumar, Saumya K. Patel, Ravi G. Kapopara, 1, 2 1, 2 Yogesh T. Jasrai, and Himanshu A. Pandya Department of Bioinformatics, Applied Botany Center, University School of Sciences, Gujarat University, Ahmedabad 380 009, India Department of Botany, University School of Sciences, Gujarat University, Ahmedabad 380 009, India Correspondence should be addressed to Yogesh T. Jasrai, yjasrai@yahoo.com Received 24 August 2012; Revised 7 November 2012; Accepted 8 November 2012 Academic Editor: Akhilesh Kumar Tyagi Copyright © 2012 Sivakumar Prasanth Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tomato leaf curl disease (ToLCD) is manifested by yellowing of leaf lamina with upward leaf curl, leaf distortion, shrinking of the leaf surface, and stunted plant growth caused by tomato leaf curl virus (ToLCV). In the present study, using computational methods we explored the evolutionary and molecular prospects of viral coat protein derived from an isolate of Vadodara district, Gujarat (ToLCGV-[Vad]), India. We found that the amino acids in coat protein required for systemic infection, viral particle formation, and insect transmission to host cells were conserved amongst Indian strains. Phylogenetic studies on Indian ToLCV coat proteins showed evolutionary compatibility with other viral taxa. Modeling of coat protein revealed a topology similar to characteristic Geminate viral particle consisting of antiparallel β-barrel motif with N-terminus α-helix. The molecular interaction of coat protein with the viral DNA required for encapsidation and nuclear shuttling was investigated through sequence- and structure-based approaches. We further emphasized the role of loops in coat protein structure as molecular recognition interface. 1. Introduction Infected plants seem healthy but develop symptoms leading to enormous economic loss [2]. Tomato leaf curl virus (ToLCV) is one of the most devas- In Indian subcontinent, ToLCV is a major problem for tating causal agents of tomato (Solanum lycopersicum)crop tomato-growing regions as several reports on new strains which had emerged causing damage and encroaching new have been documented including New Delhi, Lucknow, Ban- areas in tropical and subtropical continents every year. Plant- galore, Varanasi, Mirzapur, Vadodara, and so forth and posed infecting geminiviruses belong to the family Geminiviridae a threat to crop productivity [6]. Indian ToLCV isolates are in which Begomovirus is one among the genera possessing mostly monopartite (DNA-A) in nature with few isolates both mono- and bipartite genomes that infect especially possessing bipartite (DNA-A and DNA-B) genome organi- dicotyledonous plant species [1]. The disease is marked by zation such as tomato leaf curl New Delhi virus (ToLCNDV) symptoms such as yellowing of leaf lamina with upward leaf and tomato leaf curl Palampur virus (ToLCPalV) [7]. Both curl as well as distortion, reduction in internodes, new leaves DNA-A and DNA-B are single-stranded (ss) DNA genomes size reduction, wrinkle facade, stunted growth, and dissemi- of approximately 2.7 kb size and encode viral factors essential nation of flower from plant before onset of fruiting. ToLCV for viral replication, encapsidation, transmission, and sys- is primarily transmitted by sweet potato whitefly (Bemisia temic spread [8]. Jyothsna et al. 2012 reported tomato leaf tabaci) and silver leaf whitefly (also called Biotype B; Bemisia curl Gujarat virus (ToLCGV) possesses monopartite genome argentifolii). Whiteflies harboring virus can nonspecifically and is infectious expressing systemic symptoms in Nicotiana infect a wide spectrum of plant crops and weeds includ- benthamiana and tomato [9]. An increased symptom severity ing eggplant, potato, tobacco, pepper, and common bean. and shortened incubation period required for symptom 2 International Journal of Plant Genomics expression was noticed when ToLCGV was coinoculated CD- Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/ with betasatellite of tomato yellow leaf curl virus Thailand wrpsb.cgi, database searched: CDD v3.03–42251 PSSMs) (TYLCTHB) resulted in yellow mottling [9]. The molecular [17], PSI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) relationship of ToLCGV-[Vad], an isolate from Vadodara [18], and Pfam (http://pfam.sanger.ac.uk/)[19]. CD-Search district of Gujarat, with other strains revealed that it belongs is a NCBI’s interface to search Conserved Domain Database to Old World Begomoviruses and established a closely related (CDD) which utilizes RPS-BLAST (Reverse-PSI-BLAST; a cluster with other North Indian strains including ToLCGV- variant of PSI-BLAST) to scan a set of precalculated position (Varanasi)-[Var] and ToLCGV-(Mirzapur)-[Mir] based on specific scoring matrices (PSSMs) using a protein query. the DNA-A sequence alignment [10]. PSI-BLAST (position-specific-iterated BLAST) uses initial The measure “breeding for resistance” conceptualizes the matches to query sequence to build scoring matrix and introduction of resistance genes found in wild tomato species appends additional matches to the matrix by an iterative into tomato cultivars to develop resistance against diseases. search method in order to detect remote homologs. Pfam Kunik et al. 1994 demonstrated that tomato plants trans- designates protein family by HMM (Hidden Markov Model)- formed with TYLCV coat protein were found to be virus- based search (default settings were chosen and Pfam-A signi- resistant [11]. In India, Agrobacterium tumefaciens mediated ficant matches were only considered) over known protein transformation of coat protein gene was carried out to family classifiers. develop ToLCV tolerant/resistant transgenic tomato plants under glass house conditions [12]. Transgenic tomato plants containing cucumber mosaic virus coat protein gene was 2.3. Analysis of Nuclear Localization Signal and Its Prediction. also successfully transformed [13]. An asymmetric synergism Nuclear localization signals (NLSs) were predicted using and virulent pseudorecombinant between ToLCNDV and cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/)[20]ascoat ToLCGV was reported by Chakraborty et al. 2008 and found protein that is known to be karyophilic [21]. cNLS Mapper enhanced pathogenicity when tested in N. benthamiana, N. is a computer program that predicts NLS by activity-based tabacum, and S. lycopersicum [14]. An evidence for natu- profile search and an additivity-based motif scoring function ral recombination was observed between tomato leaf curl in different classes of importin-α/β pathway-specific NLS. Bangalore virus (ToLCBV), ToLCBV [Ban 5], and ToLCBV Theprediction wasmadewithascorecut-off of 5.0 and [Kolar] and examined the possibility of recombination searched for both mono- and bipartite NLSs with a long between strains/species that coexist within the same geo- linker (13–20 amino acid length) as ToLCGV possesses graphical location [15]. Hence, tremendous consideration mono-bipartite genome organization [6]. Classic NLS typ- should be given to study the biological and molecular ically rich in basic amino acids such as lysine and arginine, properties of this newly emerging causal agent. the counts of basic amino acids was performed manually In the present study, we examined the evolutionary in the above predicted NLSs. Comparison with literature- and molecular prospects of ToLCGV-[Vad] coat protein. reported NLS specific to BR1 nuclear export family was Sequence analysis of coat protein revealed that amino acids carried out to examine the pattern of nuclear localiza- essential for systemic infection, viral particle formation, and tion. This was achieved using pairwise sequence alignment insect transmission to host cells were evolutionarily com- using EMBOSS Stretcher (http://www.ebi.ac.uk/Tools/psa/ patible when compared to non-Indian isolates giving clues of emboss stretcher/)[22] with a representative family protein evolutionary conservativeness. Further, molecular modeling member (BR1 nuclear shuttle protein from squash leaf curl of coat protein provided a topology similar to characteristic virus(SqLCV); NCBI accession No. NP 047247.2) against Geminate viral particle. Electronic properties of coat protein the coat protein of study. Both sequences were aligned facilitated its interaction with viral DNA with the loop using EBLOSUM62 scoring matrix with a gap opening and element acting as molecular recognition interface which is extending penalty of 12 and 2. one of the major findings of the present study. 2.4. Multiple Sequence Alignment and Phylogenetic Analysis 2. Materials and Methods of Coat Proteins. Coat protein sequences of Indian strains were used for the analysis of multiple sequence alignment 2.1. Protein Sequence Retrieval. The coat protein of ToLCGV- (MSA). MSA was performed using EBI ClustalW program [Vad] (accession no. AAL78666.1) was retrieved from NCBI (http://www.ebi.ac.uk/Tools/msa/clustalw2/)[23]inwhich database (http://www.ncbi.nlm.nih.gov/)[16]. Coat pro- the sequences were aligned pairwise initially (gap open teins from Indian strains (Bangalore-CAA88227.1, Bangalore penalty = 10, gap extension penalty = 0.1, matrix = Gonnet) (Ban4)-AAD51286.1, Bangalore (Ban5)-AAK19178.1, Ban- and then the best local pairs (gap open penalty = 10, gap galore (Kolar)-AAL26553.1, Varanasi-AAO25668.1, Kelloo- extension penalty = 0.20, matrix = Gonnet) were clustered by AAM21566.1, Karnataka-AAB08929.1, New Delhi (Mild)- Neighbour-joining (NJ) technique. Subsequently, an align- AAA92817.1, and Lucknow-CAA76209.1) were also obtained ment file in ClustalW format was generated and specified as for multiple sequence alignment and phylogenetic analysis. input to draw phylogenetic tree using Phylip version 3.68 package (http://evolution.genetics.washington.edu/phylip/) 2.2. Protein Family Classification. The family of coat protein [24]. NJ algorithm was used to draw tree with inclusion of was studied using a combination of programs, namely, NCBI branch length. International Journal of Plant Genomics 3 2.5. Structure Modeling of Coat Protein. Sequence-based structure was predicted using three independent prediction similarity searching was initially executed in NCBI nonre- programs (PSI-PRED (http://bioinf.cs.ucl.ac.uk/psipred/), dundant (NR) database using BLASTp program (http://blast SSPro (http://download.igb.uci.edu/sspro4.html)and Jnet .ncbi.nlm.nih.gov/Blast.cgi)[25] with default settings to find (http://www.compbio.dundee.ac.uk/Software/JNet/jnet.html)) a close homolog with known 3D protein structure infor- and a consensus prediction was made consequently. This mation is known. Similarly, BLAST based homolog search profile and secondary structure were then scanned against in RCSB Protein Data Bank (PDB; http://www.rcsb.org/ the fold library using a profile-profile alignment algorithm to pdb/home/home.do)[26] was also carried out. Both of these generate 3D models. Followed by a reconstruction procedure procedures yielded no close homologs. So, we opted to model in the last stage, side-chains are packed using rotamer library the coat protein using homology domain modeling and and best models (selected based on confidence and sequence remote-based homology modeling. coverage) were returned. 2.6. Disorderness Prediction. In order to characterize regions 2.9. Energy Minimization and Structure Validation of Models. of sequences in coat protein which can be efficiently mod- The modeled structures were energy minimized using a eled, disorderness prediction was made. Disordered residue utility in Tripos Benchware 3D Explorer (academic ver- was identified using DISOPRED server (http://bioinf.cs.ucl sion; Tripos: A Certara company, http://www.tripos.com/) .ac.uk/disopred/)[27] with a filter threshold of 5% and a [30] with AMBER7 force field. Modeled structures were false positive threshold of 2%. The disorderness is predicted then validated for structure correctness and stereochemistry by scanning the available sequence records in the PDB and using Ramachandran plot [31] from RAMPAGE server then matches the electron density map to identify the missing (http://mordred.bioc.cam.ac.uk/∼rapper/rampage.php)[32]. coordinates. As a result, atomic coordinates of such amino Based on the percentage of favourness and frequency of acids will not be available for modeling of the protein and has outliers, the models were selected and used for further the greater possibility of producing an irregular loop region analysis. in the modeled coat protein. Thus, manual search (only at the N-and C-terminals) for disordered sequence window in 2.10. Prediction of Sequence- and Structure-Based DNA Bind- DISOPRED predictions was performed with the intention of ing Properties. In vitro studies showed that coat proteins excluding the corresponding region for modeling the coat from Geminiviridae family bind nonspecifically with both protein. It was also ensured that disordered residue reported ss- and ds-viral DNA [21, 33, 34]. To elucidate the role of in the intervening sequence positions was left out so that the DNA binding abilities of coat protein, sequence- and structure model did not possess any gaps. structure-based approaches were used. BindN (http://bioinfo .ggc.org/bindn/) employs support vector machines (SVMs) 2.7. Homology Domain Modeling. Robetta server (http:// trained from data instances such as side chain pK value, robetta.bakerlab.org/) was used for modeling the coat pro- hydrophobicity index, and molecular mass of an amino acid tein in which Ginzu, a hierarchical domain parsing and [35]. A specificity of 80% with a filter threshold of 5% was modeling protocol was adopted [28]. The input sequence chosen to avoid overwhelmed predictions. PreDs (http:// (coat protein excluded with disorderness) was initially pre-s.protein.osaka-u.ac.jp/preds/)makes useofmolecular searched using BLAST, PSI-BLAST, FFAS03 (http://ffas surface to evaluate electrostatic potential, local, and global .burnham.org/), and 3D-Jury (http://meta.bioinfo.pl/)to curvatures of the PDB queried structure to predict potential obtain information on homologous regions which are dsDNA binding sites [36]. The modeled coat protein was then modeled with their comparative modeling protocol. defined as input with validation chosen from scoring func- Unassigned (i.e., nonhomologous as identified in the first tions. stage) regions were then parsed to model as domain linkers using a combination of approaches, namely, HMMER search 2.11. Viral DNA Structure Modeling and Docking with (http://hmmer.janelia.org/) over Pfam-A database and an Coat Protein. Canonical viral DNA was modeled using 3D- MSA (produced from initial PSI-BLAST results) based search DART (3DNA-Driven DNA Analysis and Rebuilding Tool; over NCBI NR database. Subsequently, K Sync alignment http://haddock.chem.uu.nl/dna/dna.php) web service with method was utilized to predict elements that are obligated to default introduction of parameters for bends (roll, tilt, and the fold to produce a single default alignment by dynamic twist) [37]. It uses 3DNA “fiber” module to generate canon- programming. Best five models were generated after loop ical DNA structure and “find pair” and “analyze” modules modeling, domain assembly, and side-chain packing. to produce a corresponding base pair (step) parameter file. The parameter file was used to set up local and global 2.8. Remote-Based Homology Modeling. Efforts were also bends in the DNA structure file which are then remodeled carried out to predict structure of coat protein using remote- finally using “rebuild” component to return PDB formatted based homology modeling approach with the help of protein DNA structure file. The docking phase was carried out using homology/analogy recognition engine (Phyre) version HADDOCK (High Ambiguity Driven biomolecular DOCK- 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/)[29]. In the first ing; http://haddock.chem.uu.nl/)program [38] with the step, profile was constructed using five iterations of PSI- modeled coat protein and ds-viral DNA as inputs. Residues BLAST against NR sequence database. The query secondary encompassed in a DNA binding region predicted by PreDs 4 International Journal of Plant Genomics ToLCGV-[Vad] 18 91 CDD: pfam00844 71 Pfam HMM search ToLCGV-[Vad] 92 170 CDD: pfam00844 72 Pfam HMM search ToLCGV-[Vad] 171 CDD: pfam00844 152 Pfam HMM search ToLCGV-[Vad] 256 CDD: pfam00844 232 Pfam HMM search Figure 1: Sequence alignment of ToLCGV-[Vad] coat protein with nuclear export factor of BR1 family (Pfam entry: 00844 recovered from NCBI CDD) and HMM profile of the geminivirus coat protein. and utilized molecular surface (computed with a probe with a Parea of greater than 250 A was specified as active site residues whereas passive residues were automatically radius of 1.4 A) to set up the dielectric boundary. The resul- defined around the active site which forms the boundary of tant electrostatic potential grid map in data explorer (dx) the DNA-binding region. This specification was introduced format was recovered and specified as input to PyMol version to enhance the conformational search space for docking 2.5 program (academic version; Schrodinger LLC) [41]to simulation as well as to avoid blindfold docking experiments. view the PBEQ electrostatic map. Definition of residues takes the form of experimental data which were converted into ambiguous interaction restraints 3. Results and Discussion (AIRs) in order to generate topology of the structures sub- sequently. The docking procedure consists of three stages: an 3.1. Prediction of Protein Family of Coat Protein. The protein energy minimization in a rigid-body manner, a semiflexible family of ToLCV coat protein was predicted using a combina- refinement in torsional space, and a final refinement in tion of programs. NCBI CD-Search using protein sequence explicit solvent. After execution of each of these stages, the revealed that it belongs to Gemini-coat protein superfamily resultant structures are scored and ranked and the best fitted (Pfam entry: pfam00844, accession no. Q8QYY9). Upon structures are employed in next stages. The best docked carefully examining the sequence alignment generated (E- conformation can be obtained (usually clustered at the top) value: 5.53e-100; bit score: 290.36) with SqLCV BR1 nuclear by inspecting the HADDOCK score which is a summation of shuttle protein, it was studied that ToLCGV belongs to intermolecular energies, namely, van der Waals (vdW), elec- nuclear export factor BR1 family (Figure 1). BR1 is a ssDNA trostatic (Elec), desolvation (Dsolv) and AIRS together with binding protein that shuttles between the nucleus and cyto- buried surface area (BSA): rigid-body score = 1.0 ∗ Elec + plasm in plant cells [33]. 1.0 ∗ vdW−0.05 ∗ BSA + 1.0 ∗ Dsolv + 1.0 ∗ AIR; final PSI-BLAST sequence hit (PSI-BLAST threshold: 0.005 score = 1.0 ∗ Elec + 1.0 ∗ vdW + 1.0 ∗ Dsolv + 1.0 ∗ AIR. maximum iterations: 7; E-value: 1e-105; bit score: 383; sequence coverage in alignment: 99.64%) with a capsid pro- 2.12. Generation of Electrostatic Potential Map for Docked tein of Begomovirus taxa (UniRef90 P03560; tomato golden Structures. The influence of electrostatics for enabling DNA- mosaic virus) was observed. Further, sequence-based query protein interaction was studied using continuum Poisson- over Pfam-A (Pfam-B not chosen as we focused on obtaining Boltzmann (PB) electrostatic approach. It was achieved highly curated data) database produced a result similar to by PBEQ-Solver (PBEQuation-Solver; http://www.charmm- NCBI CD-Search. This HMM-based search provided an gui.org/?doc=input/pbeqsolver)[39] for which PQR files alignment with an E-value of 2.3e-87 and bit score of 292.1 were required as molecular inputs. Hence, the docked com- (Figure 1). Manual inspection of PubMed references in the plexes in PDB format were converted into PQR format using pfam00844 entry in NCBI CDD disclosed that coat proteins PDB2PQR server (http://nbcr-222.ucsd.edu/pdb2pqr 1.8/) of Geminiviridae family binds ss- and ds-viral DNA in [40]. PQR format embodies the replacement of occupancy vitro [42]. For instance, TYLCV coat protein [43], maize column in a PDB file (“P”) with the atomic charge (“Q”) and streak virus (MSV) coat protein [21], SqLCV nuclear shuttle the temperature factor column with the atomic radius (“R”). protein [44], and bean dwarf mosaic geminivirus(BDMV) The inputted PDB file was subjected to following struc- movement protein [34] have the same function of binding tural manipulations: rebuilding missing heavy atoms, build- which helps them to establish infection by nuclear shuttling ing and optimizing hydrogens and assignment of atomic of viral DNA across cell boundaries. Besides, coat protein charges and radii based on force field parameters from also possesses binding function necessary for encapsidation CHARMM22 (selected option), AMBER99 or PARSE. All the of viral DNA. It is well known that the genomic component PB calculations on PBEQ-Solver were performed in a coarse DNA-B in bipartite Begomovirus such as ToLCNDV encodes ˚ ˚ grid space (before focusing = 1.5 A and after focusing = 1.0 A) two movement proteins, namely, nuclear shuttle protein International Journal of Plant Genomics 5 (a) Predicted and experimental NLS SqLCV 25 MSV 1 TYLCV 1 ToLCV 1 (b) Crucial amino acids TYLCV-Sic 125 155 TYLCV-SicRv 125 155 TYLCV-Sar 125 Indian wild ToLCV strains Bangalore 125 155 Bangalore (Ban4) 125 155 Bangalore (Ban5) 125 Bangalore (Kolar) 125 155 Karnataka 125 155 New Delhi (Mild) 125 155 Lucknow 125 Gujarat (Vadodara) 125 155 Varanasi 125 155 Kelloo 125 Figure 2: (a) MSA of predicted and experimental NLS of selected geminivirus coat proteins. (b) MSA of coat proteins from Indian and non-Indian strains corresponding to sequence region of interest containing the key amino acids (highlighted in larger fonts) required for systemic infection, viral particle formation, and insect transmission. (NSP) and cell-to-cell movement protein (MP) that direct scientific literatures related to BR1 nuclear export family the viral genome to the cortical cytoplasm and across the bar- in order to infer the predictions made. Pairwise sequence rier of the cell wall for infection multiplication [45]. On the alignment of NLS region from MSV and ToLCGV-[Vad] coat other hand, monopartite Begomovirus including ToLCGV proteins resulted in an identity and similarity percentage of [9] produces coat protein and other associated proteins to 25% and 33.3% with a score of −6whereas TYLCVand alleviate movement inside the host while coat protein acts as ToLCGV-[Vad] yielded an identity and similarity percentage nuclear shuttler facilitating import and export of DNA [46]. of 51.7% and 58.6% with a score of 49. This pairwise Therefore, it is anticipated that coat protein of ToLCGV may alignment suggested that ToLCV coat protein is much more also function as nuclear shuttler. conserved with TYLCV rather than SqLCV [3](identity and similarity = 15%, score = −20), a representative protein member of BR1 family (pfam00844) in Pfam database. 3.2. Prediction of NLS in the Coat Protein Sequence. Muta- genesis study on MSV coat protein [4]and TYLCV[5] NLS region resulted in the cytoplasmic accumulation of the 3.3. MSA and Phylogenetic Analysis of Indian Strains. ToLCV mutant protein. Thus, ToLCV coat protein must possess a coat protein sequences from Indian strains were retrieved NLS region in its sequence in order to be translocated to from NCBI database to construct MSA in order to study nucleus. A NLS signal was predicted with a score 10.2 by the amino acids crucial in conserved domain and responsible cNLS Mapper in the coat protein N-terminal with a for systemic infection, viral particle formation, and insect composition of 20 amino acids (predicted bipartite NLS: transmission. Norris et al. 1998 reported that a functional MSKRPADMLIFTPASKVRRR, predicted monopartite NLS: coat protein having amino acids in the following sequence none). positions, namely, Pro/Gln129, Gln/His134, and Glu/Asp152 The occurrence of basic amino acids in the predicted NLS on TYLCV isolates is essential for correct assembly of virions showed that lysine and arginine constituted 2 and 4 counts and transmission by the insect vector [47]. These key residues which proposed to have a classic NLS pattern (Table 1) were identified by B. tabaci transmissibility studies in the and can be comparable to experimentally identified TYLCV field isolates of TYLCV-Sic (Sicily), TYLCV-Sar (Sardinia), coat protein NLS [5](Figure 2(a)). Despite the impressive and TYLCV-SicRv (engineered mutant of Sicily) [47]. Exam- number of receptor-cargo interactions that have been stud- ination of corresponding positions in our MSA cluster ied, the prediction of NLSs in candidate proteins remains revealed that Lys129, Ser/Thr134, and Asp151 (instead of extremely difficult. So, we step forwarded our search in 152nd position as a result of single residue deletion) were 6 International Journal of Plant Genomics Table 1: Known and predicted NLS pattern of BR1 nuclear export family. Organism Frequency of lysine residues Frequency of arginine residues Predicted NLS pattern KRSYGAARGDDRRRP SqLCV coat protein 15 (Sanderfoot et al., 1996 [3]) MSTSKRKRGDDSNWSKRVTKKKPS MSV coat protein 63 (Liu et al., 1999 [4]) MSKRPGDIIISTPVSKVRRRLNFDSPYSS TYLCV coat protein 24 (Kunik et al., 1998 [5]) MSKRPADMLIFTPASKVRRR ToLCGV-[Vad] 24 (predicted by cNLS Mapper) conserved amongst Indian strains in comparison to non- was predicted as loop region with a variety of secondary Indian isolates and are found to be wild-type. The com- structure prediction programs including PSI-Pred, GORIV, parison of chemical properties of the template with that of and so forth. So, we decided to exclude NLS signal from the MSA showed that a positively charged amino acid (Lys129) protein sequence for modeling due to the consideration of was identified in the uncharged polar (Gln129) position. disordered profile and the increased possibility of generating The second important residue (Ser/Thr134 in replacement loop geometry. with Gln/His134) was conserved in terms of polarity while a negatively charged residue (Glu/Asp152; Asp151) was pre- 3.5. Structure Modeling of Coat Protein. No close template served in the third crucial position (Figure 2(b)). This amino was obtained in an attempt to find structurally known acids combination (Lys129, Ser/Thr134, and Asp121) is also homolog of ToLCV coat protein using BLASTp and sequence conserved in coat proteins among different wild-type viruses, based BLAST search in PDB with default settings. Therefore, namely, tomato golden mosaic virus, tomato mottle virus- we decided to use homology domain modeling using Robetta [Florida], sinaloatomato leaf curl virus, tomato leaf crumple program and remote-based homology modeling using Phyre virus, taino tomato mottle virus, abutilon mosaic virus- program. [Hawaii], bean golden mosaic virus-[Brazil], SqLCV and After evaluation of predictions related to secondary papaya leaf curl virus [47]. structure features, Ginzu, a domain parser of Robetta A phylogenetic tree based on NJ algorithm was con- modeled two domains with a sequence span of 1–59 and 60– structed using Phylip version 3.68 to study the sequence 235 using Pfam and HHSearch as sources. K∗Sync alignment conservativeness among Indian strains. Surprisingly, coat method was employed subsequently to generate structurally proteins characteristic from districts, namely, Vadodara, good scoring decoys followed by loop modeling, domain Varanasi, and Kelloo were clustered in a node with a branch assembly, and side-chain packing. This procedure yielded length of 0.167. It should be noted that these members were five models. Remote-based homology modeling using Phyre representing different states in the Northern India contrast- provided a model based on the Nucleoplasmin-like/viral pro- ing to other members which were sufficiently diverged to tein (viral coat and capsid proteins) as folding unit (PDB ID: each other. Besides the fact that Bangalore isolates were con- 2BUK chain A; structure of satellite tobacco necrosis virus served among each other, they were distinct with one of the after crystallographic refinement at 2.5 A resolution) [48] state member, Karnataka with a length of 0.011. Isolates from derived from satellite virus with a confidence of 97.3. Fortu- New Delhi and Lucknow were conserved as expected in terms nately, Robetta used the same structural template for models of area nearness (Figure 3). The key amino acids required generation. Phyre provided a list of other models sorted by for biochemical functions were indeed conserved amongst its confidence level were found to only range from 8.03 to each other with respect to the comparison using MSA made 23.9. above. 3.4. Disorderness and Their Link with Predicted NLS. Dis- 3.6. Selection of Best Scoring Models. A total of six coat pro- orderness was predicted in the coat protein to identify the tein models (five obtained from Robetta program and one sequence regions that cannot be modeled efficiently with the from Phyre program) were subjected to energy minimization protein modeling procedures adopted by us. This scrutiny with AMBER7 force field and 250 as maximum number of was taken to eliminate the loop region in the sequence evaluations using minimize energy module engineered in terminals. Disorder profile produced with 5% filter threshold Tripos Benchware 3D Explorer. Energy minimized structures showed that a window with sequence positions from 1 to 50 were then validated using stereochemistry check with the was scattered with peaks demonstrating residue disorderness help of Ramachandran plot. The Robetta model (energy (Figure 4). This region corresponds to NLS in the N-ter- minimized to −2682.00 Kcal/mol) was chosen based on minal. There exists a relationship between the predicted NLS the residue occurrence of more than 95% estimated by and the disorderness as the corresponding sequence position summing up favorable and allowed regions (Figure 5)in ϕ-ψ International Journal of Plant Genomics 7 0.02 0.016 ToLCV Karnataka 0.011 0.013 ToLCV Lucknow 0.037 ToLCV New Delhi (Mild) ToLCV Kelloo 0.167 ToLCV Varanasi ToLCV Vadodara 0.011 ToLCV Bangalore (Kolar) ToLCV Bangalore (Ban5) 0.062 0.021 ToLCV Bangalore (Ban4) ToLCV Bangalore 0.014 Figure 3: Phylogenetic tree of ToLCV coat protein representing closeness among Indian strains. Disordered profile region (two helices expected at the N-terminal) and eleven β- strands (only eight were expected instead of eleven) as it was 0.6 not complied with characteristic Geminate viral particle. So, we discarded these models for further analysis. 0.4 3.7. Resemblance to Geminate Viral Particle. The modeled 0.2 ToLCV coat protein was compared with the structure of the MSV Geminate particle, a member of Geminiviridae fam- ily determined using cryo-electron microscopy and three- 0 50 100 150 200 250 dimensional image reconstruction methods [49]. The mod- Sequence positions eled protein possessed an N-terminal helix with an eight- Filter stranded antiparallel β-barrel motif characteristic of Gem- 5% filter threshold inate particle. The β-barrel motif is a dominant structural Output unit in all ssDNA virus structures that have been determined to atomic resolution. Unlike the model generated by Zhang Figure 4: Disordered profile of ToLCV coat protein with disorder- et al. 2001 [49], our Robetta model has 7 reliable and 1 ness in the sequence positions 1–50 which contains predicted NLS. short (total 8 strands) antiparallel β-strands and 2 helices at the N-terminal instead of 1 in comparison to Zhang’s Gemi- nate model. We also noticed 1 helix at the intervening region core areas and the presence of only one outlier (Glu204) (Figure 6). We expected that this additional accumulation whereas the best scored Phyre model was discarded due to of secondary structural elements is beyond the evolution the loose packing of loop regions and its close resemblance and may be an additional procurement in the dsDNA virus to Robetta model (root mean squared deviation (RMSD): structures in disparity to ssDNA virus structures due to con- 20.8578 A over 1041 matched atoms). There is one more servational pressures as described by Zhang et al. 2001 [49] reason to unconsider the Phyre model as the N-terminus was or due to the insertional sequences or it may be due to the constituted with loop elements, two helices in the intervening loop geometry as it was undistinguished by the present Disorder probability 8 International Journal of Plant Genomics GLU204 Ψ 0 −180 −1800 180 General/pre-pro/proline favoured Glycine favoured General/pre-pro/proline allowed Glycine allowed Figure 5: Best scored ToLCV Robetta model showing one outlier (Glu204) on Ramachandran plot. (a) (b) Figure 6: Developed ToLCV models using homology domain modeling (Robetta program; (a)) and remote-based homology modeling (Phyre program; (b)). H-Helix, E-Extended β-strands. programs due to the nonavailability of experimental atomic reveal the crucial amino acids involved in DNA interaction, information. sequence- and structure-based approaches were utilized. BindN predicted spatially distributed residues as component of DNA binding interface with 74 amino acids and 28.90% 3.8. Sequence and Structure-Based DNA Binding Properties. contribution (Figure 7). We expected that this widespread ToLCV must possess DNA binding properties for accom- distribution of DNA binding residues will come together plishing several cellular functions such as nuclear targeting of during protein folding and will interact with viral DNA. viral DNA, encapsidation of viral DNA, systemic infection, Thus, we step forwarded to identify those prominent amino viral particle formation, insect transmission, and correct acids using our generated ToLCV coat model. PreDs revealed assembly of virions as experimentally studied in other mem- loop regions as potential DNA binding region accompanied bers of the family, namely, MSV, SqLCV, BDMV, and so forth. with all those amino acids predicted by BindN. This predic- Interestingly, the coat proteins bind both ss- and ds-viral tion was validated by inspecting the scoring functions such as DNA in sequence independent fashion [21, 33, 34]. To International Journal of Plant Genomics 9 Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Figure 7: Sequence-based DNA binding properties of ToLCV coat protein. DNA binding residues are shown in red text. Pscore and Parea. Pscore is an indicator for the ratio of the predicted area possessing maximum value while Parea represents area of the predicted ds-DNA binding region on the protein surface. We achieved 0.31 as Pscore (threshold: 2 2 ˚ ˚ >0.12) and 2102.26 A (threshold: >250 A ) as Parea, respec- tively (Figure 8). The TYLCV coat protein gets imported into the plant and insect cells nuclei via using its N- terminal NLS [5]. It is also been proposed that TYLCV coat protein functions as BR1 protein facilitating DNA trafficking across cell boundaries (0.31, 2102.26) and demonstrated that the coat protein binds DNA cooper- atively [43]. It is also highlighted that TYLCV coat protein may also aid in protecting the transported coat protein-DNA complex from intracellular nucleolytic degradation as this complex was highly resistant to in vitro S1 nuclease activity [43]. In MSV coat protein, the DNA binding domain was mapped to the N-terminal 104 amino acids inclusive of NLS [21]. Immuno-electron microscopy revealed that DNA 0 0.12 0.5 binding domain between residues 5 and 22 suggested that Pscore this region could be involved in transporting geminivirus coat protein towards nuclei [50]. We predicted that certain Figure 8: Structure based DNA binding properties of ToLCV coat N-terminal amino acids of ToLCV coat protein scored a con- protein having reliable Pscore and Parea. fidence of 6–9 indicating DNA binding properties (Figure 7). Besides, structural analysis of modeled ToLCV indicated that N-terminal residues contributing to loop secondary struc- ture form the major element in interacting with viral DNA interface of coat protein was found to be the loop region as described below. whereas major groove was the molecular interface of viral DNA in which the best conformers were sampled (Figure 9). 3.9. Viral DNA Structure Modeling and Docking with Coat The intermolecular energies (unit in KJ/mol) obtained are Protein. We developed a canonical ds-viral DNA using 3D- as follows: vdW: −66.9 ± 5.5, Elec: 827.4 ± 87.8, Dsolv: DART with generalized geometrical constraints to explore 125.7± 11.7, AIR: 947.0± 50.09, and BSA: 2099.6± 137.9. We the interaction with coat protein. Molecular docking was also observed that RMSD of overall lowest-energy structure performed using HADDOCK program with predicted DNA was 4.3 ± 3.0 with respect to structures in different clusters. binding region as active site. Best scoring clusters were sorted The internal energy in apo form (free molecules) was based on HADDOCK score. The most reliable (top) cluster 278722.00 KJ/mol whereas in bound form (DNA-protein having four similar docked conformations (HADDOCK complex) was 15494.00 KJ/mol and the binding energy was score: −12.0 ± 11.0) were recovered. The DNA binding predicted as −264139.00 KJ/mol. Parea 10 International Journal of Plant Genomics Vi Virral dsDN aldsDNA A To ToL LC CV V c co oat at pr prot otei ein n Viral DNA ToLCV coat protein −1 Figure 10: Electrostatic potential map of ToLCV coat protein- viral dsDNA complex. Neutral patch of coat protein interacts with negatively charged DNA. Figure 9: The binding mode of ToLCV coat protein with viral dsDNA where loop element of protein and major groove of DNA acting as interfaces. nucleus is associated with resistance whereas large aggregates leading to infection susceptibility [53]. We highlighted sequence regions in ToLCV coat protein possessing both 3.10. Electrostatic Interaction of Coat Protein. DNA binding DNA binding properties and the functional amino acids interface predicted by PreDs was found to be electrostat- combination essential for virulence and these regions of ically favored as its prediction was principally based on interest can be targeted by developing siRNA. In addition, electrostatic potential, local, and global curvatures present these molecular properties of ToLCV coat protein can be on the DNA surface. HADDOCK revealed coat protein’s accounted in developing Begomovirus resistant-engineered loop topology as its molecular interface unit. DNA-protein plants. interaction is predominantly influenced by electrostatics and can be efficiently studied using adaptive Poisson-Boltzmann solver (APBS) approach. The docked conformation was 4. Conclusion charged appropriately based on physiological environment ToLCV coat protein possesses DNA binding properties to with preprocessing using PDB2PQR and all PB calculations function similar to BR1 nuclear shuttler. The amino acid were carried out using PBEQ-Solver in a coarse grid space. combinations crucial for virulence were investigated through Electrostatic grid map analyzed using PyMol showed that MSA and the evolutionary relationship was traced by neutral patches in coat protein were found to interact with phylogenetic analysis which indicated that the Indian strains viral DNA (Figure 10). We expected that an isosurface of are closely related in the context of geographical locations. positive patch will tend to interact with negatively charged The predicted NLS of ToLCV coat protein shares more simi- viral DNA. Upon manual inspection of charged clusters in larity with experimentally known TYLCV coat protein NLS. the coat protein, the positive charged clusters were cornered Molecular modeling represented ToLCV coat protein as in surface with significantly low negative patches and a Geminate viral particle. Further, sequence- and structure- greater deal of positive regions corresponded to β-barrel based approaches identified that ToLCV coat protein through motif. We ruled out the requirement of positive patch its loop topology interacts with viral DNA as surface comple- contributing to DNA interaction as geometrical flexibility of mentarity proven to be the major promoting factor followed loop region in the coat protein having neutrality was much by electrostatic interaction. We anticipate the conserved favored rather than charge-charge attraction. This view was region of ToLCV coat protein prominent for DNA binding further inferred by the best docked conformation in which and functional sequence pattern can be targeted by RNA electrostatic energy term was investigated and found to be the interference to develop disease-resistant transgenic tomato major intermolecular descriptor representing interaction. plants. Zrachya et al. 2007 reported that TYLCV coat protein could be targeted by small interfering ds RNAs (siRNAs) derived from intron-hairpin RNA construct to develop Acknowledgments disease resistant transgenic tomato cultivars and showed its potential in N. benthamiana transient assays [51]. Similar S. P. Kumar acknowledges generous support from DST, New studies targeting against antisense replicase gene (AC1) in Delhi as Innovation in Science Pursuit for Inspired Research ToLCV helped in developing trait-stable transgenics [52]. (INSPIRE) Fellowship. The authors would like to thank three Besides, the midsized aggregation of coat protein inside anonymous reviewers for their fruitful suggestions. International Journal of Plant Genomics 11 References [15] N. Kirthi, S. P. Maiya, M. R. N. Murthy, and H. S. Savithri, “Evidence for recombination among the Tomato leaf curl virus [1] B. 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Evolutionary and Molecular Aspects of Indian Tomato Leaf Curl Virus Coat Protein

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Copyright © 2012 Sivakumar Prasanth Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2012, Article ID 417935, 12 pages doi:10.1155/2012/417935 Research Article Evolutionary and Molecular Aspects of Indian Tomato Leaf Curl Virus Coat Protein 1 1 1 Sivakumar Prasanth Kumar, Saumya K. Patel, Ravi G. Kapopara, 1, 2 1, 2 Yogesh T. Jasrai, and Himanshu A. Pandya Department of Bioinformatics, Applied Botany Center, University School of Sciences, Gujarat University, Ahmedabad 380 009, India Department of Botany, University School of Sciences, Gujarat University, Ahmedabad 380 009, India Correspondence should be addressed to Yogesh T. Jasrai, yjasrai@yahoo.com Received 24 August 2012; Revised 7 November 2012; Accepted 8 November 2012 Academic Editor: Akhilesh Kumar Tyagi Copyright © 2012 Sivakumar Prasanth Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tomato leaf curl disease (ToLCD) is manifested by yellowing of leaf lamina with upward leaf curl, leaf distortion, shrinking of the leaf surface, and stunted plant growth caused by tomato leaf curl virus (ToLCV). In the present study, using computational methods we explored the evolutionary and molecular prospects of viral coat protein derived from an isolate of Vadodara district, Gujarat (ToLCGV-[Vad]), India. We found that the amino acids in coat protein required for systemic infection, viral particle formation, and insect transmission to host cells were conserved amongst Indian strains. Phylogenetic studies on Indian ToLCV coat proteins showed evolutionary compatibility with other viral taxa. Modeling of coat protein revealed a topology similar to characteristic Geminate viral particle consisting of antiparallel β-barrel motif with N-terminus α-helix. The molecular interaction of coat protein with the viral DNA required for encapsidation and nuclear shuttling was investigated through sequence- and structure-based approaches. We further emphasized the role of loops in coat protein structure as molecular recognition interface. 1. Introduction Infected plants seem healthy but develop symptoms leading to enormous economic loss [2]. Tomato leaf curl virus (ToLCV) is one of the most devas- In Indian subcontinent, ToLCV is a major problem for tating causal agents of tomato (Solanum lycopersicum)crop tomato-growing regions as several reports on new strains which had emerged causing damage and encroaching new have been documented including New Delhi, Lucknow, Ban- areas in tropical and subtropical continents every year. Plant- galore, Varanasi, Mirzapur, Vadodara, and so forth and posed infecting geminiviruses belong to the family Geminiviridae a threat to crop productivity [6]. Indian ToLCV isolates are in which Begomovirus is one among the genera possessing mostly monopartite (DNA-A) in nature with few isolates both mono- and bipartite genomes that infect especially possessing bipartite (DNA-A and DNA-B) genome organi- dicotyledonous plant species [1]. The disease is marked by zation such as tomato leaf curl New Delhi virus (ToLCNDV) symptoms such as yellowing of leaf lamina with upward leaf and tomato leaf curl Palampur virus (ToLCPalV) [7]. Both curl as well as distortion, reduction in internodes, new leaves DNA-A and DNA-B are single-stranded (ss) DNA genomes size reduction, wrinkle facade, stunted growth, and dissemi- of approximately 2.7 kb size and encode viral factors essential nation of flower from plant before onset of fruiting. ToLCV for viral replication, encapsidation, transmission, and sys- is primarily transmitted by sweet potato whitefly (Bemisia temic spread [8]. Jyothsna et al. 2012 reported tomato leaf tabaci) and silver leaf whitefly (also called Biotype B; Bemisia curl Gujarat virus (ToLCGV) possesses monopartite genome argentifolii). Whiteflies harboring virus can nonspecifically and is infectious expressing systemic symptoms in Nicotiana infect a wide spectrum of plant crops and weeds includ- benthamiana and tomato [9]. An increased symptom severity ing eggplant, potato, tobacco, pepper, and common bean. and shortened incubation period required for symptom 2 International Journal of Plant Genomics expression was noticed when ToLCGV was coinoculated CD- Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/ with betasatellite of tomato yellow leaf curl virus Thailand wrpsb.cgi, database searched: CDD v3.03–42251 PSSMs) (TYLCTHB) resulted in yellow mottling [9]. The molecular [17], PSI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) relationship of ToLCGV-[Vad], an isolate from Vadodara [18], and Pfam (http://pfam.sanger.ac.uk/)[19]. CD-Search district of Gujarat, with other strains revealed that it belongs is a NCBI’s interface to search Conserved Domain Database to Old World Begomoviruses and established a closely related (CDD) which utilizes RPS-BLAST (Reverse-PSI-BLAST; a cluster with other North Indian strains including ToLCGV- variant of PSI-BLAST) to scan a set of precalculated position (Varanasi)-[Var] and ToLCGV-(Mirzapur)-[Mir] based on specific scoring matrices (PSSMs) using a protein query. the DNA-A sequence alignment [10]. PSI-BLAST (position-specific-iterated BLAST) uses initial The measure “breeding for resistance” conceptualizes the matches to query sequence to build scoring matrix and introduction of resistance genes found in wild tomato species appends additional matches to the matrix by an iterative into tomato cultivars to develop resistance against diseases. search method in order to detect remote homologs. Pfam Kunik et al. 1994 demonstrated that tomato plants trans- designates protein family by HMM (Hidden Markov Model)- formed with TYLCV coat protein were found to be virus- based search (default settings were chosen and Pfam-A signi- resistant [11]. In India, Agrobacterium tumefaciens mediated ficant matches were only considered) over known protein transformation of coat protein gene was carried out to family classifiers. develop ToLCV tolerant/resistant transgenic tomato plants under glass house conditions [12]. Transgenic tomato plants containing cucumber mosaic virus coat protein gene was 2.3. Analysis of Nuclear Localization Signal and Its Prediction. also successfully transformed [13]. An asymmetric synergism Nuclear localization signals (NLSs) were predicted using and virulent pseudorecombinant between ToLCNDV and cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/)[20]ascoat ToLCGV was reported by Chakraborty et al. 2008 and found protein that is known to be karyophilic [21]. cNLS Mapper enhanced pathogenicity when tested in N. benthamiana, N. is a computer program that predicts NLS by activity-based tabacum, and S. lycopersicum [14]. An evidence for natu- profile search and an additivity-based motif scoring function ral recombination was observed between tomato leaf curl in different classes of importin-α/β pathway-specific NLS. Bangalore virus (ToLCBV), ToLCBV [Ban 5], and ToLCBV Theprediction wasmadewithascorecut-off of 5.0 and [Kolar] and examined the possibility of recombination searched for both mono- and bipartite NLSs with a long between strains/species that coexist within the same geo- linker (13–20 amino acid length) as ToLCGV possesses graphical location [15]. Hence, tremendous consideration mono-bipartite genome organization [6]. Classic NLS typ- should be given to study the biological and molecular ically rich in basic amino acids such as lysine and arginine, properties of this newly emerging causal agent. the counts of basic amino acids was performed manually In the present study, we examined the evolutionary in the above predicted NLSs. Comparison with literature- and molecular prospects of ToLCGV-[Vad] coat protein. reported NLS specific to BR1 nuclear export family was Sequence analysis of coat protein revealed that amino acids carried out to examine the pattern of nuclear localiza- essential for systemic infection, viral particle formation, and tion. This was achieved using pairwise sequence alignment insect transmission to host cells were evolutionarily com- using EMBOSS Stretcher (http://www.ebi.ac.uk/Tools/psa/ patible when compared to non-Indian isolates giving clues of emboss stretcher/)[22] with a representative family protein evolutionary conservativeness. Further, molecular modeling member (BR1 nuclear shuttle protein from squash leaf curl of coat protein provided a topology similar to characteristic virus(SqLCV); NCBI accession No. NP 047247.2) against Geminate viral particle. Electronic properties of coat protein the coat protein of study. Both sequences were aligned facilitated its interaction with viral DNA with the loop using EBLOSUM62 scoring matrix with a gap opening and element acting as molecular recognition interface which is extending penalty of 12 and 2. one of the major findings of the present study. 2.4. Multiple Sequence Alignment and Phylogenetic Analysis 2. Materials and Methods of Coat Proteins. Coat protein sequences of Indian strains were used for the analysis of multiple sequence alignment 2.1. Protein Sequence Retrieval. The coat protein of ToLCGV- (MSA). MSA was performed using EBI ClustalW program [Vad] (accession no. AAL78666.1) was retrieved from NCBI (http://www.ebi.ac.uk/Tools/msa/clustalw2/)[23]inwhich database (http://www.ncbi.nlm.nih.gov/)[16]. Coat pro- the sequences were aligned pairwise initially (gap open teins from Indian strains (Bangalore-CAA88227.1, Bangalore penalty = 10, gap extension penalty = 0.1, matrix = Gonnet) (Ban4)-AAD51286.1, Bangalore (Ban5)-AAK19178.1, Ban- and then the best local pairs (gap open penalty = 10, gap galore (Kolar)-AAL26553.1, Varanasi-AAO25668.1, Kelloo- extension penalty = 0.20, matrix = Gonnet) were clustered by AAM21566.1, Karnataka-AAB08929.1, New Delhi (Mild)- Neighbour-joining (NJ) technique. Subsequently, an align- AAA92817.1, and Lucknow-CAA76209.1) were also obtained ment file in ClustalW format was generated and specified as for multiple sequence alignment and phylogenetic analysis. input to draw phylogenetic tree using Phylip version 3.68 package (http://evolution.genetics.washington.edu/phylip/) 2.2. Protein Family Classification. The family of coat protein [24]. NJ algorithm was used to draw tree with inclusion of was studied using a combination of programs, namely, NCBI branch length. International Journal of Plant Genomics 3 2.5. Structure Modeling of Coat Protein. Sequence-based structure was predicted using three independent prediction similarity searching was initially executed in NCBI nonre- programs (PSI-PRED (http://bioinf.cs.ucl.ac.uk/psipred/), dundant (NR) database using BLASTp program (http://blast SSPro (http://download.igb.uci.edu/sspro4.html)and Jnet .ncbi.nlm.nih.gov/Blast.cgi)[25] with default settings to find (http://www.compbio.dundee.ac.uk/Software/JNet/jnet.html)) a close homolog with known 3D protein structure infor- and a consensus prediction was made consequently. This mation is known. Similarly, BLAST based homolog search profile and secondary structure were then scanned against in RCSB Protein Data Bank (PDB; http://www.rcsb.org/ the fold library using a profile-profile alignment algorithm to pdb/home/home.do)[26] was also carried out. Both of these generate 3D models. Followed by a reconstruction procedure procedures yielded no close homologs. So, we opted to model in the last stage, side-chains are packed using rotamer library the coat protein using homology domain modeling and and best models (selected based on confidence and sequence remote-based homology modeling. coverage) were returned. 2.6. Disorderness Prediction. In order to characterize regions 2.9. Energy Minimization and Structure Validation of Models. of sequences in coat protein which can be efficiently mod- The modeled structures were energy minimized using a eled, disorderness prediction was made. Disordered residue utility in Tripos Benchware 3D Explorer (academic ver- was identified using DISOPRED server (http://bioinf.cs.ucl sion; Tripos: A Certara company, http://www.tripos.com/) .ac.uk/disopred/)[27] with a filter threshold of 5% and a [30] with AMBER7 force field. Modeled structures were false positive threshold of 2%. The disorderness is predicted then validated for structure correctness and stereochemistry by scanning the available sequence records in the PDB and using Ramachandran plot [31] from RAMPAGE server then matches the electron density map to identify the missing (http://mordred.bioc.cam.ac.uk/∼rapper/rampage.php)[32]. coordinates. As a result, atomic coordinates of such amino Based on the percentage of favourness and frequency of acids will not be available for modeling of the protein and has outliers, the models were selected and used for further the greater possibility of producing an irregular loop region analysis. in the modeled coat protein. Thus, manual search (only at the N-and C-terminals) for disordered sequence window in 2.10. Prediction of Sequence- and Structure-Based DNA Bind- DISOPRED predictions was performed with the intention of ing Properties. In vitro studies showed that coat proteins excluding the corresponding region for modeling the coat from Geminiviridae family bind nonspecifically with both protein. It was also ensured that disordered residue reported ss- and ds-viral DNA [21, 33, 34]. To elucidate the role of in the intervening sequence positions was left out so that the DNA binding abilities of coat protein, sequence- and structure model did not possess any gaps. structure-based approaches were used. BindN (http://bioinfo .ggc.org/bindn/) employs support vector machines (SVMs) 2.7. Homology Domain Modeling. Robetta server (http:// trained from data instances such as side chain pK value, robetta.bakerlab.org/) was used for modeling the coat pro- hydrophobicity index, and molecular mass of an amino acid tein in which Ginzu, a hierarchical domain parsing and [35]. A specificity of 80% with a filter threshold of 5% was modeling protocol was adopted [28]. The input sequence chosen to avoid overwhelmed predictions. PreDs (http:// (coat protein excluded with disorderness) was initially pre-s.protein.osaka-u.ac.jp/preds/)makes useofmolecular searched using BLAST, PSI-BLAST, FFAS03 (http://ffas surface to evaluate electrostatic potential, local, and global .burnham.org/), and 3D-Jury (http://meta.bioinfo.pl/)to curvatures of the PDB queried structure to predict potential obtain information on homologous regions which are dsDNA binding sites [36]. The modeled coat protein was then modeled with their comparative modeling protocol. defined as input with validation chosen from scoring func- Unassigned (i.e., nonhomologous as identified in the first tions. stage) regions were then parsed to model as domain linkers using a combination of approaches, namely, HMMER search 2.11. Viral DNA Structure Modeling and Docking with (http://hmmer.janelia.org/) over Pfam-A database and an Coat Protein. Canonical viral DNA was modeled using 3D- MSA (produced from initial PSI-BLAST results) based search DART (3DNA-Driven DNA Analysis and Rebuilding Tool; over NCBI NR database. Subsequently, K Sync alignment http://haddock.chem.uu.nl/dna/dna.php) web service with method was utilized to predict elements that are obligated to default introduction of parameters for bends (roll, tilt, and the fold to produce a single default alignment by dynamic twist) [37]. It uses 3DNA “fiber” module to generate canon- programming. Best five models were generated after loop ical DNA structure and “find pair” and “analyze” modules modeling, domain assembly, and side-chain packing. to produce a corresponding base pair (step) parameter file. The parameter file was used to set up local and global 2.8. Remote-Based Homology Modeling. Efforts were also bends in the DNA structure file which are then remodeled carried out to predict structure of coat protein using remote- finally using “rebuild” component to return PDB formatted based homology modeling approach with the help of protein DNA structure file. The docking phase was carried out using homology/analogy recognition engine (Phyre) version HADDOCK (High Ambiguity Driven biomolecular DOCK- 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/)[29]. In the first ing; http://haddock.chem.uu.nl/)program [38] with the step, profile was constructed using five iterations of PSI- modeled coat protein and ds-viral DNA as inputs. Residues BLAST against NR sequence database. The query secondary encompassed in a DNA binding region predicted by PreDs 4 International Journal of Plant Genomics ToLCGV-[Vad] 18 91 CDD: pfam00844 71 Pfam HMM search ToLCGV-[Vad] 92 170 CDD: pfam00844 72 Pfam HMM search ToLCGV-[Vad] 171 CDD: pfam00844 152 Pfam HMM search ToLCGV-[Vad] 256 CDD: pfam00844 232 Pfam HMM search Figure 1: Sequence alignment of ToLCGV-[Vad] coat protein with nuclear export factor of BR1 family (Pfam entry: 00844 recovered from NCBI CDD) and HMM profile of the geminivirus coat protein. and utilized molecular surface (computed with a probe with a Parea of greater than 250 A was specified as active site residues whereas passive residues were automatically radius of 1.4 A) to set up the dielectric boundary. The resul- defined around the active site which forms the boundary of tant electrostatic potential grid map in data explorer (dx) the DNA-binding region. This specification was introduced format was recovered and specified as input to PyMol version to enhance the conformational search space for docking 2.5 program (academic version; Schrodinger LLC) [41]to simulation as well as to avoid blindfold docking experiments. view the PBEQ electrostatic map. Definition of residues takes the form of experimental data which were converted into ambiguous interaction restraints 3. Results and Discussion (AIRs) in order to generate topology of the structures sub- sequently. The docking procedure consists of three stages: an 3.1. Prediction of Protein Family of Coat Protein. The protein energy minimization in a rigid-body manner, a semiflexible family of ToLCV coat protein was predicted using a combina- refinement in torsional space, and a final refinement in tion of programs. NCBI CD-Search using protein sequence explicit solvent. After execution of each of these stages, the revealed that it belongs to Gemini-coat protein superfamily resultant structures are scored and ranked and the best fitted (Pfam entry: pfam00844, accession no. Q8QYY9). Upon structures are employed in next stages. The best docked carefully examining the sequence alignment generated (E- conformation can be obtained (usually clustered at the top) value: 5.53e-100; bit score: 290.36) with SqLCV BR1 nuclear by inspecting the HADDOCK score which is a summation of shuttle protein, it was studied that ToLCGV belongs to intermolecular energies, namely, van der Waals (vdW), elec- nuclear export factor BR1 family (Figure 1). BR1 is a ssDNA trostatic (Elec), desolvation (Dsolv) and AIRS together with binding protein that shuttles between the nucleus and cyto- buried surface area (BSA): rigid-body score = 1.0 ∗ Elec + plasm in plant cells [33]. 1.0 ∗ vdW−0.05 ∗ BSA + 1.0 ∗ Dsolv + 1.0 ∗ AIR; final PSI-BLAST sequence hit (PSI-BLAST threshold: 0.005 score = 1.0 ∗ Elec + 1.0 ∗ vdW + 1.0 ∗ Dsolv + 1.0 ∗ AIR. maximum iterations: 7; E-value: 1e-105; bit score: 383; sequence coverage in alignment: 99.64%) with a capsid pro- 2.12. Generation of Electrostatic Potential Map for Docked tein of Begomovirus taxa (UniRef90 P03560; tomato golden Structures. The influence of electrostatics for enabling DNA- mosaic virus) was observed. Further, sequence-based query protein interaction was studied using continuum Poisson- over Pfam-A (Pfam-B not chosen as we focused on obtaining Boltzmann (PB) electrostatic approach. It was achieved highly curated data) database produced a result similar to by PBEQ-Solver (PBEQuation-Solver; http://www.charmm- NCBI CD-Search. This HMM-based search provided an gui.org/?doc=input/pbeqsolver)[39] for which PQR files alignment with an E-value of 2.3e-87 and bit score of 292.1 were required as molecular inputs. Hence, the docked com- (Figure 1). Manual inspection of PubMed references in the plexes in PDB format were converted into PQR format using pfam00844 entry in NCBI CDD disclosed that coat proteins PDB2PQR server (http://nbcr-222.ucsd.edu/pdb2pqr 1.8/) of Geminiviridae family binds ss- and ds-viral DNA in [40]. PQR format embodies the replacement of occupancy vitro [42]. For instance, TYLCV coat protein [43], maize column in a PDB file (“P”) with the atomic charge (“Q”) and streak virus (MSV) coat protein [21], SqLCV nuclear shuttle the temperature factor column with the atomic radius (“R”). protein [44], and bean dwarf mosaic geminivirus(BDMV) The inputted PDB file was subjected to following struc- movement protein [34] have the same function of binding tural manipulations: rebuilding missing heavy atoms, build- which helps them to establish infection by nuclear shuttling ing and optimizing hydrogens and assignment of atomic of viral DNA across cell boundaries. Besides, coat protein charges and radii based on force field parameters from also possesses binding function necessary for encapsidation CHARMM22 (selected option), AMBER99 or PARSE. All the of viral DNA. It is well known that the genomic component PB calculations on PBEQ-Solver were performed in a coarse DNA-B in bipartite Begomovirus such as ToLCNDV encodes ˚ ˚ grid space (before focusing = 1.5 A and after focusing = 1.0 A) two movement proteins, namely, nuclear shuttle protein International Journal of Plant Genomics 5 (a) Predicted and experimental NLS SqLCV 25 MSV 1 TYLCV 1 ToLCV 1 (b) Crucial amino acids TYLCV-Sic 125 155 TYLCV-SicRv 125 155 TYLCV-Sar 125 Indian wild ToLCV strains Bangalore 125 155 Bangalore (Ban4) 125 155 Bangalore (Ban5) 125 Bangalore (Kolar) 125 155 Karnataka 125 155 New Delhi (Mild) 125 155 Lucknow 125 Gujarat (Vadodara) 125 155 Varanasi 125 155 Kelloo 125 Figure 2: (a) MSA of predicted and experimental NLS of selected geminivirus coat proteins. (b) MSA of coat proteins from Indian and non-Indian strains corresponding to sequence region of interest containing the key amino acids (highlighted in larger fonts) required for systemic infection, viral particle formation, and insect transmission. (NSP) and cell-to-cell movement protein (MP) that direct scientific literatures related to BR1 nuclear export family the viral genome to the cortical cytoplasm and across the bar- in order to infer the predictions made. Pairwise sequence rier of the cell wall for infection multiplication [45]. On the alignment of NLS region from MSV and ToLCGV-[Vad] coat other hand, monopartite Begomovirus including ToLCGV proteins resulted in an identity and similarity percentage of [9] produces coat protein and other associated proteins to 25% and 33.3% with a score of −6whereas TYLCVand alleviate movement inside the host while coat protein acts as ToLCGV-[Vad] yielded an identity and similarity percentage nuclear shuttler facilitating import and export of DNA [46]. of 51.7% and 58.6% with a score of 49. This pairwise Therefore, it is anticipated that coat protein of ToLCGV may alignment suggested that ToLCV coat protein is much more also function as nuclear shuttler. conserved with TYLCV rather than SqLCV [3](identity and similarity = 15%, score = −20), a representative protein member of BR1 family (pfam00844) in Pfam database. 3.2. Prediction of NLS in the Coat Protein Sequence. Muta- genesis study on MSV coat protein [4]and TYLCV[5] NLS region resulted in the cytoplasmic accumulation of the 3.3. MSA and Phylogenetic Analysis of Indian Strains. ToLCV mutant protein. Thus, ToLCV coat protein must possess a coat protein sequences from Indian strains were retrieved NLS region in its sequence in order to be translocated to from NCBI database to construct MSA in order to study nucleus. A NLS signal was predicted with a score 10.2 by the amino acids crucial in conserved domain and responsible cNLS Mapper in the coat protein N-terminal with a for systemic infection, viral particle formation, and insect composition of 20 amino acids (predicted bipartite NLS: transmission. Norris et al. 1998 reported that a functional MSKRPADMLIFTPASKVRRR, predicted monopartite NLS: coat protein having amino acids in the following sequence none). positions, namely, Pro/Gln129, Gln/His134, and Glu/Asp152 The occurrence of basic amino acids in the predicted NLS on TYLCV isolates is essential for correct assembly of virions showed that lysine and arginine constituted 2 and 4 counts and transmission by the insect vector [47]. These key residues which proposed to have a classic NLS pattern (Table 1) were identified by B. tabaci transmissibility studies in the and can be comparable to experimentally identified TYLCV field isolates of TYLCV-Sic (Sicily), TYLCV-Sar (Sardinia), coat protein NLS [5](Figure 2(a)). Despite the impressive and TYLCV-SicRv (engineered mutant of Sicily) [47]. Exam- number of receptor-cargo interactions that have been stud- ination of corresponding positions in our MSA cluster ied, the prediction of NLSs in candidate proteins remains revealed that Lys129, Ser/Thr134, and Asp151 (instead of extremely difficult. So, we step forwarded our search in 152nd position as a result of single residue deletion) were 6 International Journal of Plant Genomics Table 1: Known and predicted NLS pattern of BR1 nuclear export family. Organism Frequency of lysine residues Frequency of arginine residues Predicted NLS pattern KRSYGAARGDDRRRP SqLCV coat protein 15 (Sanderfoot et al., 1996 [3]) MSTSKRKRGDDSNWSKRVTKKKPS MSV coat protein 63 (Liu et al., 1999 [4]) MSKRPGDIIISTPVSKVRRRLNFDSPYSS TYLCV coat protein 24 (Kunik et al., 1998 [5]) MSKRPADMLIFTPASKVRRR ToLCGV-[Vad] 24 (predicted by cNLS Mapper) conserved amongst Indian strains in comparison to non- was predicted as loop region with a variety of secondary Indian isolates and are found to be wild-type. The com- structure prediction programs including PSI-Pred, GORIV, parison of chemical properties of the template with that of and so forth. So, we decided to exclude NLS signal from the MSA showed that a positively charged amino acid (Lys129) protein sequence for modeling due to the consideration of was identified in the uncharged polar (Gln129) position. disordered profile and the increased possibility of generating The second important residue (Ser/Thr134 in replacement loop geometry. with Gln/His134) was conserved in terms of polarity while a negatively charged residue (Glu/Asp152; Asp151) was pre- 3.5. Structure Modeling of Coat Protein. No close template served in the third crucial position (Figure 2(b)). This amino was obtained in an attempt to find structurally known acids combination (Lys129, Ser/Thr134, and Asp121) is also homolog of ToLCV coat protein using BLASTp and sequence conserved in coat proteins among different wild-type viruses, based BLAST search in PDB with default settings. Therefore, namely, tomato golden mosaic virus, tomato mottle virus- we decided to use homology domain modeling using Robetta [Florida], sinaloatomato leaf curl virus, tomato leaf crumple program and remote-based homology modeling using Phyre virus, taino tomato mottle virus, abutilon mosaic virus- program. [Hawaii], bean golden mosaic virus-[Brazil], SqLCV and After evaluation of predictions related to secondary papaya leaf curl virus [47]. structure features, Ginzu, a domain parser of Robetta A phylogenetic tree based on NJ algorithm was con- modeled two domains with a sequence span of 1–59 and 60– structed using Phylip version 3.68 to study the sequence 235 using Pfam and HHSearch as sources. K∗Sync alignment conservativeness among Indian strains. Surprisingly, coat method was employed subsequently to generate structurally proteins characteristic from districts, namely, Vadodara, good scoring decoys followed by loop modeling, domain Varanasi, and Kelloo were clustered in a node with a branch assembly, and side-chain packing. This procedure yielded length of 0.167. It should be noted that these members were five models. Remote-based homology modeling using Phyre representing different states in the Northern India contrast- provided a model based on the Nucleoplasmin-like/viral pro- ing to other members which were sufficiently diverged to tein (viral coat and capsid proteins) as folding unit (PDB ID: each other. Besides the fact that Bangalore isolates were con- 2BUK chain A; structure of satellite tobacco necrosis virus served among each other, they were distinct with one of the after crystallographic refinement at 2.5 A resolution) [48] state member, Karnataka with a length of 0.011. Isolates from derived from satellite virus with a confidence of 97.3. Fortu- New Delhi and Lucknow were conserved as expected in terms nately, Robetta used the same structural template for models of area nearness (Figure 3). The key amino acids required generation. Phyre provided a list of other models sorted by for biochemical functions were indeed conserved amongst its confidence level were found to only range from 8.03 to each other with respect to the comparison using MSA made 23.9. above. 3.4. Disorderness and Their Link with Predicted NLS. Dis- 3.6. Selection of Best Scoring Models. A total of six coat pro- orderness was predicted in the coat protein to identify the tein models (five obtained from Robetta program and one sequence regions that cannot be modeled efficiently with the from Phyre program) were subjected to energy minimization protein modeling procedures adopted by us. This scrutiny with AMBER7 force field and 250 as maximum number of was taken to eliminate the loop region in the sequence evaluations using minimize energy module engineered in terminals. Disorder profile produced with 5% filter threshold Tripos Benchware 3D Explorer. Energy minimized structures showed that a window with sequence positions from 1 to 50 were then validated using stereochemistry check with the was scattered with peaks demonstrating residue disorderness help of Ramachandran plot. The Robetta model (energy (Figure 4). This region corresponds to NLS in the N-ter- minimized to −2682.00 Kcal/mol) was chosen based on minal. There exists a relationship between the predicted NLS the residue occurrence of more than 95% estimated by and the disorderness as the corresponding sequence position summing up favorable and allowed regions (Figure 5)in ϕ-ψ International Journal of Plant Genomics 7 0.02 0.016 ToLCV Karnataka 0.011 0.013 ToLCV Lucknow 0.037 ToLCV New Delhi (Mild) ToLCV Kelloo 0.167 ToLCV Varanasi ToLCV Vadodara 0.011 ToLCV Bangalore (Kolar) ToLCV Bangalore (Ban5) 0.062 0.021 ToLCV Bangalore (Ban4) ToLCV Bangalore 0.014 Figure 3: Phylogenetic tree of ToLCV coat protein representing closeness among Indian strains. Disordered profile region (two helices expected at the N-terminal) and eleven β- strands (only eight were expected instead of eleven) as it was 0.6 not complied with characteristic Geminate viral particle. So, we discarded these models for further analysis. 0.4 3.7. Resemblance to Geminate Viral Particle. The modeled 0.2 ToLCV coat protein was compared with the structure of the MSV Geminate particle, a member of Geminiviridae fam- ily determined using cryo-electron microscopy and three- 0 50 100 150 200 250 dimensional image reconstruction methods [49]. The mod- Sequence positions eled protein possessed an N-terminal helix with an eight- Filter stranded antiparallel β-barrel motif characteristic of Gem- 5% filter threshold inate particle. The β-barrel motif is a dominant structural Output unit in all ssDNA virus structures that have been determined to atomic resolution. Unlike the model generated by Zhang Figure 4: Disordered profile of ToLCV coat protein with disorder- et al. 2001 [49], our Robetta model has 7 reliable and 1 ness in the sequence positions 1–50 which contains predicted NLS. short (total 8 strands) antiparallel β-strands and 2 helices at the N-terminal instead of 1 in comparison to Zhang’s Gemi- nate model. We also noticed 1 helix at the intervening region core areas and the presence of only one outlier (Glu204) (Figure 6). We expected that this additional accumulation whereas the best scored Phyre model was discarded due to of secondary structural elements is beyond the evolution the loose packing of loop regions and its close resemblance and may be an additional procurement in the dsDNA virus to Robetta model (root mean squared deviation (RMSD): structures in disparity to ssDNA virus structures due to con- 20.8578 A over 1041 matched atoms). There is one more servational pressures as described by Zhang et al. 2001 [49] reason to unconsider the Phyre model as the N-terminus was or due to the insertional sequences or it may be due to the constituted with loop elements, two helices in the intervening loop geometry as it was undistinguished by the present Disorder probability 8 International Journal of Plant Genomics GLU204 Ψ 0 −180 −1800 180 General/pre-pro/proline favoured Glycine favoured General/pre-pro/proline allowed Glycine allowed Figure 5: Best scored ToLCV Robetta model showing one outlier (Glu204) on Ramachandran plot. (a) (b) Figure 6: Developed ToLCV models using homology domain modeling (Robetta program; (a)) and remote-based homology modeling (Phyre program; (b)). H-Helix, E-Extended β-strands. programs due to the nonavailability of experimental atomic reveal the crucial amino acids involved in DNA interaction, information. sequence- and structure-based approaches were utilized. BindN predicted spatially distributed residues as component of DNA binding interface with 74 amino acids and 28.90% 3.8. Sequence and Structure-Based DNA Binding Properties. contribution (Figure 7). We expected that this widespread ToLCV must possess DNA binding properties for accom- distribution of DNA binding residues will come together plishing several cellular functions such as nuclear targeting of during protein folding and will interact with viral DNA. viral DNA, encapsidation of viral DNA, systemic infection, Thus, we step forwarded to identify those prominent amino viral particle formation, insect transmission, and correct acids using our generated ToLCV coat model. PreDs revealed assembly of virions as experimentally studied in other mem- loop regions as potential DNA binding region accompanied bers of the family, namely, MSV, SqLCV, BDMV, and so forth. with all those amino acids predicted by BindN. This predic- Interestingly, the coat proteins bind both ss- and ds-viral tion was validated by inspecting the scoring functions such as DNA in sequence independent fashion [21, 33, 34]. To International Journal of Plant Genomics 9 Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Sequence: Prediction: Confidence: Figure 7: Sequence-based DNA binding properties of ToLCV coat protein. DNA binding residues are shown in red text. Pscore and Parea. Pscore is an indicator for the ratio of the predicted area possessing maximum value while Parea represents area of the predicted ds-DNA binding region on the protein surface. We achieved 0.31 as Pscore (threshold: 2 2 ˚ ˚ >0.12) and 2102.26 A (threshold: >250 A ) as Parea, respec- tively (Figure 8). The TYLCV coat protein gets imported into the plant and insect cells nuclei via using its N- terminal NLS [5]. It is also been proposed that TYLCV coat protein functions as BR1 protein facilitating DNA trafficking across cell boundaries (0.31, 2102.26) and demonstrated that the coat protein binds DNA cooper- atively [43]. It is also highlighted that TYLCV coat protein may also aid in protecting the transported coat protein-DNA complex from intracellular nucleolytic degradation as this complex was highly resistant to in vitro S1 nuclease activity [43]. In MSV coat protein, the DNA binding domain was mapped to the N-terminal 104 amino acids inclusive of NLS [21]. Immuno-electron microscopy revealed that DNA 0 0.12 0.5 binding domain between residues 5 and 22 suggested that Pscore this region could be involved in transporting geminivirus coat protein towards nuclei [50]. We predicted that certain Figure 8: Structure based DNA binding properties of ToLCV coat N-terminal amino acids of ToLCV coat protein scored a con- protein having reliable Pscore and Parea. fidence of 6–9 indicating DNA binding properties (Figure 7). Besides, structural analysis of modeled ToLCV indicated that N-terminal residues contributing to loop secondary struc- ture form the major element in interacting with viral DNA interface of coat protein was found to be the loop region as described below. whereas major groove was the molecular interface of viral DNA in which the best conformers were sampled (Figure 9). 3.9. Viral DNA Structure Modeling and Docking with Coat The intermolecular energies (unit in KJ/mol) obtained are Protein. We developed a canonical ds-viral DNA using 3D- as follows: vdW: −66.9 ± 5.5, Elec: 827.4 ± 87.8, Dsolv: DART with generalized geometrical constraints to explore 125.7± 11.7, AIR: 947.0± 50.09, and BSA: 2099.6± 137.9. We the interaction with coat protein. Molecular docking was also observed that RMSD of overall lowest-energy structure performed using HADDOCK program with predicted DNA was 4.3 ± 3.0 with respect to structures in different clusters. binding region as active site. Best scoring clusters were sorted The internal energy in apo form (free molecules) was based on HADDOCK score. The most reliable (top) cluster 278722.00 KJ/mol whereas in bound form (DNA-protein having four similar docked conformations (HADDOCK complex) was 15494.00 KJ/mol and the binding energy was score: −12.0 ± 11.0) were recovered. The DNA binding predicted as −264139.00 KJ/mol. Parea 10 International Journal of Plant Genomics Vi Virral dsDN aldsDNA A To ToL LC CV V c co oat at pr prot otei ein n Viral DNA ToLCV coat protein −1 Figure 10: Electrostatic potential map of ToLCV coat protein- viral dsDNA complex. Neutral patch of coat protein interacts with negatively charged DNA. Figure 9: The binding mode of ToLCV coat protein with viral dsDNA where loop element of protein and major groove of DNA acting as interfaces. nucleus is associated with resistance whereas large aggregates leading to infection susceptibility [53]. We highlighted sequence regions in ToLCV coat protein possessing both 3.10. Electrostatic Interaction of Coat Protein. DNA binding DNA binding properties and the functional amino acids interface predicted by PreDs was found to be electrostat- combination essential for virulence and these regions of ically favored as its prediction was principally based on interest can be targeted by developing siRNA. In addition, electrostatic potential, local, and global curvatures present these molecular properties of ToLCV coat protein can be on the DNA surface. HADDOCK revealed coat protein’s accounted in developing Begomovirus resistant-engineered loop topology as its molecular interface unit. DNA-protein plants. interaction is predominantly influenced by electrostatics and can be efficiently studied using adaptive Poisson-Boltzmann solver (APBS) approach. The docked conformation was 4. Conclusion charged appropriately based on physiological environment ToLCV coat protein possesses DNA binding properties to with preprocessing using PDB2PQR and all PB calculations function similar to BR1 nuclear shuttler. The amino acid were carried out using PBEQ-Solver in a coarse grid space. combinations crucial for virulence were investigated through Electrostatic grid map analyzed using PyMol showed that MSA and the evolutionary relationship was traced by neutral patches in coat protein were found to interact with phylogenetic analysis which indicated that the Indian strains viral DNA (Figure 10). We expected that an isosurface of are closely related in the context of geographical locations. positive patch will tend to interact with negatively charged The predicted NLS of ToLCV coat protein shares more simi- viral DNA. Upon manual inspection of charged clusters in larity with experimentally known TYLCV coat protein NLS. the coat protein, the positive charged clusters were cornered Molecular modeling represented ToLCV coat protein as in surface with significantly low negative patches and a Geminate viral particle. Further, sequence- and structure- greater deal of positive regions corresponded to β-barrel based approaches identified that ToLCV coat protein through motif. We ruled out the requirement of positive patch its loop topology interacts with viral DNA as surface comple- contributing to DNA interaction as geometrical flexibility of mentarity proven to be the major promoting factor followed loop region in the coat protein having neutrality was much by electrostatic interaction. We anticipate the conserved favored rather than charge-charge attraction. This view was region of ToLCV coat protein prominent for DNA binding further inferred by the best docked conformation in which and functional sequence pattern can be targeted by RNA electrostatic energy term was investigated and found to be the interference to develop disease-resistant transgenic tomato major intermolecular descriptor representing interaction. plants. Zrachya et al. 2007 reported that TYLCV coat protein could be targeted by small interfering ds RNAs (siRNAs) derived from intron-hairpin RNA construct to develop Acknowledgments disease resistant transgenic tomato cultivars and showed its potential in N. benthamiana transient assays [51]. Similar S. P. Kumar acknowledges generous support from DST, New studies targeting against antisense replicase gene (AC1) in Delhi as Innovation in Science Pursuit for Inspired Research ToLCV helped in developing trait-stable transgenics [52]. (INSPIRE) Fellowship. The authors would like to thank three Besides, the midsized aggregation of coat protein inside anonymous reviewers for their fruitful suggestions. International Journal of Plant Genomics 11 References [15] N. Kirthi, S. P. Maiya, M. R. N. Murthy, and H. S. Savithri, “Evidence for recombination among the Tomato leaf curl virus [1] B. 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