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1 UK Journal of Pharmaceutical and Biosciences Vol. 4(2), 58-62, 2016 RESEARCH ARTICLE UK Journal of Pharmaceutical and Biosciences Available at ISSN: AB Initio Structural Analysis on Envelope (E) Glycoprotein of Dengue Virus () Type 1-4 Venkataramanan S 1*, Noor Adurrah MFL 1, Zulhabri O 1, Kavitha K 2 1 Faculty Health and Life Sciences, Management and Science University, Shah Alam-40100, Malaysia 2 School of Bio Sciences and Technology, VIT University, Vellore , Tamilnadu, India Article Information Received 10 March 2016 Received in revised form 6 April 2016 Accepted 7 April 2016 Keywords: Dengue virus, envelope glycoprotein, B-cell epitopes, T-cell epitopes. *Corresponding Author: s_venkataramanan@msu.edu.my Mob. : xxxxxxxxxxxx Abstract Any serotypes of dengue virus (), which are, may lead to dengue infections. spread via mosquito vector, primarily Aedes aegypti and Aedes albopictus mosquitoes. is one of the most prevalent viruses, which is causing dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) that may be fatal. Currently, there are no antiviral drugs and effective vaccination available to combat infections. Major component of which plays the important role for viral binding to the receptor to induce the infection is an envelope (E) glycoprotein. The B-cell and T-cell epitopes for are not yet determined. An effective vaccine against infections should be a tetravalent vaccine that can counter-attack all four serotypes. Therefore, E glycoproteins of all four serotypes was chosen to be analyzed. Conserved regions were found in 100 sequences of each serotype. Multiple sequence alignments was done to obtain consensus sequence. Multiple bioinformatics tools were used for prediction of B-cell and T-cell epitopes based on antigenicity, hydrophilicity, beta-turn, flexibility and surface accessibility. Secondary and tertiary structures of E glycoprotein for each serotypes were predicted and aligned. Complete analysis showed that predicted epitopes were located within conserved regions, and 2 showed the highest percentage of mutation rates and had the highest similarity to 1, 3 and 4. 1 Introduction Dengue virus is a major emerging arthopod-borne pathogen causing a significant burden of disease in tropical and subtropical areas of the world. It has been reported that about millions peoples are infected by dengue every year. In addition 2.5 billion people are at risk of dengue. Most infections are either asymptomatic or result in dengue fever, a relatively mild illness 1,2. Moreover, it is estimated 1 5% of infections of dengue are life threatening form. The incidence of dengue is increasing at an alarming rate and epidemics can severely disrupt healthcare systems in developing countries. Although treatment has reduced the mortality rate, there is still an urgent need for a vaccine 3. Dengue is a Flavivirus belongs to Flaviviridae family and it is main causative agents of infectious disease and deaths in the world. It can be categorized into four distinctive serotypes, which are 1, 2, 3 and 4. spread to humans via Aedes aegypti and Aedes albopictus mosquitoes as the vectors 4. may lead to DF, DHF and DSS in humans. genome consists of a single strand RNA which encompass by an icosahedral scaffold. It is secured by an envelope layer consists of lipid 5. The genome encodes for three structural proteins (C-prM-E), followed by seven non-structural (NS) proteins (NS1 - NS2A - NS2B - NS3 - NS4A - NS4B - NS5) 6. Envelope (E) glycoprotein is located on the viral outer layer appears to be the significant part in host cell receptor attachment for viral entry and inducing protective immunity 7. Until now, there are no antiviral drugs and effective vaccination available to combat infections. It is difficult to design specific drug to treat dengue related infections as an after effect of great mutation sites within genome itself. Many efforts have been made in the search for a suitable vaccine, but the lack of an animal model and the need for a high immunogenicity vaccine against all

2 four serotypes and a low reactogenicity are posing huge challenges hydrophilicity, surface accessibility, beta-turn, flexibility and in the dengue vaccine development. antigenicity. The hydrophilic regions were predicted by using It is proposed that viral epitopes on the surface of can trigger ProtScale (Hopp & Woods method) and Antibody Epitope Prediction cellular immune responses and subsequently the development of a server (Parker method). By using Antibody Epitope Prediction server, severe disease. Therefore, these epitopes are potential targets for surface accessible, beta turn and flexible regions were determined the development of a new class of antiviral products, entry based on Emini method, Chou & Fasman method and Karplus & inhibitors. Inhibition of attachment and entry into the host cell Schulz method, respectively. The antigenic peptides were predicted can inhibit immune activation. The cellular immune response is using Antigenic Peptide Prediction server based on Kolaskar & believed to play an important role in antibody-dependent Tongaonkar method. enhancement. This is a phenomenon where cross-reacting 2.5 T-cell epitopes prediction nonneutralizing antibodies generated to the first infection will The consensus sequences of obtained from Clustal recognize a heterologous during a secondary infection with Omega were used as the data input for T-cell epitopes prediction. another serotype. This results in the proliferation of T cells and the The prediction was done by using HLAPred server. Locations of T- production of proinflammatory cytokines that have an indirect effect cell epitopes were predicted based on Human Leukocyte Antigen on the vascular endothelial cells leading to plasma leakage and (HLA) Class I, using six different alleles, which were HLA-A2, HLA- DHF. Inhibition of virus attachment is a valuable antiviral strategy A11, HLA-A24, HLA-B51, HLA-B60 and HLA-B62. because it forms the first barrier to block infection We planned to investigate interpretations of dengue virus sequence analysis based 2.6 Protein tertiary structure prediction on E glycoprotein. Web servers and other softwares were used in Prediction of the tertiary structure of E glycoprotein for each our study. serotype was done by using RaptorX server. Consensus sequences 2 Methods of all serotypes in FASTA format were used as the data input in RaptorX. 2.1 Protein sequence retrieval 2.7 Multiple structural alignment The E glycoproteins of sequences was retrieved from NCBI website. Total of 100 sequences were randomly selected for Multiple structural alignments of all serotypes was done using each serotype, in which the final total of 400 sequences are retrieved PDBeFold server. All the the four files consist of predicted tertiary from the NCBI website. The sequences were copied in FASTA structures of E glycoprotein were uploaded in the multiple format. alignment submission form. After all the files were uploaded, the submission form was submitted to obtaining the result. 2.2 Multiple sequence alignment 3 Results The E glycoprotein sequences for each serotype were aligned and compared by using Clustal Omega for identification of conserved Based on multiple sequence alignments for all serotypes, it and mutated regions. The output format was predetermined to clustal was observed that each of the serotype showed the high percentage without numbers. Other than that, the consensus sequence of each of conserved regions. The conserved regions were shown in table 1. serotype was obtained and saved to be used for further Table 1: Conserved regions of analysis. 2.3 Protein secondary structure prediction The consensus sequences of all serotypes were used as the data input in SOPMA to obtain the predicted secondary structure of E glycoprotein for each serotype. The parameters were predetermined into four states, which predict the helix, sheet, turn and coil feature of the sequence, similarity threshold of eight and 17 as the window width. 2.4 B-cell epitopes prediction The consensus sequences of obtained from Clustal Omega were used as the data input for B-cell epitopes prediction. The epitopes were predicted based on five parameters, which are Sequence Number 15-38, 62-72, , , , and , 63-83, , , , and , , , , , and , , , , , and UK J Pharm & Biosci, 2016: 4(2); 59

3 In terms of E glycoprotein secondary structure prediction, each The predicted tertiary structures of E glycoprotein for all serotype showed the highest occurrences of beta sheets and serotypes are shown in figure 2. Based on multiple structural coils compared to alpha helix and beta turn structures (Figure 1). alignment, 2 and 3 had the highest sequence identity The results of secondary structure prediction had no abrupt and similar in structure. The superposition of all serotypes are differences among all serotypes. The most possible B-cell and shown in figure 3. 2 showed the lowest RMSD score, with the T-cell epitopes for all serotypes were diagrammed in table 2 score value of and the highest Q score of (Table 4). and table 3, respectively. Figure 2: Predicted tertiary structures of E glycoprotein of Table 3: Sequence number of predicted T-cell epitopes of Sequence Position 1 50, 378, 388, 416, 432 and , 155, 232, 312, 386 and 485 Figure 1: Occurrences of alpha helix (blue), beta sheets (red), beta turns (green) and coils (green) of E glycoprotein of 1-4 Table 2: Sequence number of predicted B-cell epitopes of 3 48, 258, 310, 349, 360 and , 35, 136, 256, 260 and 390 Table 4: RMSD and Q scores of based on structural alignment Sequence Number RMSD Score Q Score , , , , , and , , , , , and , , , , , and , , , , , and UK J Pharm & Biosci, 2016: 4(2); 60

4 4 Discussions observed that majority of predicted B-cell and T-cell epitopes were located within conserved regions. Even though the E glycoprotein It is difficult to design specific drug to treat dengue related infections structure was the most variable region of, the conserved as an aftereffect of great mutation sites within genome itself. regions still can be found. The predicted tertiary structures of E Notwithstanding, numerous scientists are attempting to discover an glycoprotein of all serotypes were evaluated as good quality approach to create the drug for treatment and immunization of structure based on RaptorX server. The predicted tertiary structure of infections. The epitopes were the crucial parts to be analyzed as 2 was predicted as the most similar and closely related to the they are the ones that were interacting with the human immune other three serotypes based on the lowest RMSD and highest system to produce the antibodies, trigger the immune response and combat the infections 11 Q scores among all four serotypes. The above statement supported. the findings of Rangel et al., identified several immunodominant IgGspecific epitopes on the envelope of The epitopes are potentially relevant for the development of an effective vaccine for the dengue virus Conclusions Figure 3: Multiple structural alignment of E glycoprotein of The envelope is capable of inducing a protective immune response that is neutralizing antibody. To define epitopes which were able to induce neutralizing antibody, the envelopes were analyzed by programs. Ilyas et al 12, showed the result of 9 predicted B-cell epitopes of 3 envelope while Zhong et al 6 showed 20 predicted B-cell epitopes. It is aasumed that this peptide sequence could induce 3 neutralizing antibody and be likely to use for peptide vaccine development. Based on multiple sequence alignment results of all serotypes, a great number of polar amino acids, which are glycine (Gly), threonine (Thr) and serine (Ser) were observed in each serotype. Then, Gly), Leucine (Leu) and Valine (Val) were usually found in the conserved regions. 3 showed the highest percentage of conserved bases with the value of 85.0%, followed by 4, 1 and 2 with the value of 83.0%, 77.0% and 70.5% respectively. In contrast, 2 showed the highest percentage of mutation rates with the value of 15.2% and followed by 1 with the value of 7.3%. Both 3 and 4 showed the same percentage of mutation rates, which are 5.1%. It was observed that alpha helix, beta turn and coil structures were located beside and within the conserved regions. Therefore, it was predicted that these three structures were the ones that were maintaining the stability of E glycoprotein of all serotypes. Based on the epitopes prediction for each serotype, it was The conserved epitopes may be useful for the development of epitope-based tetravalent vaccine against infections. There should be more research work and analysis focused on the E glycoprotein structure and its interaction with antibody as it was the main component responsible in inducing the infection and triggering the immune response. Based on this study, it was concluded that most of the predicted epitopes were located within the conserved regions, stable alpha helix, beta turn and coil structures were located beside the conserved regions, and all serotypes showed the similar structural characteristics. 6 Competing interests There are no conflicts of interests. 7 Author s contributions The present research work was carried out by equal contribution of all the authors. VS, NDMFL, ZO and KK designed the experimental protocol, carried out literature review and draft the manuscript. VS participated in collection of data through experimental work. All authors read and approved the final manuscript. 8 Acknowledgements First and foremost, thanks to Allah for the completion of this thesis. Only due to his blessings I could finish my thesis. I would like to thank my beloved parents, Mr. Muhammad Faizal Lai Abdullah and Mrs. Manah Mawi, for their encouragement and support throughout my life. I would like to offer my greatest attitude to my supervisor and co-supervisor, Mr. S. Venkataramanan and Dr. Zulhabri Othman and K. Kavitha who has supported me throughout my manuscript with their patience, insightful comments, endless advice and knowledge whilst allowing me to work this project in my own way. In my daily work, I have been blessed with a cheerful group of friends and last but not least, my gratefulness to the countless support from Suhalfarina Suhaimi for the advice, stimulating discussions, sleepless nights and the fun we had in completing this manuscript. UK J Pharm & Biosci, 2016: 4(2); 61

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