Introduction

Dengue virus genome contains a positive strand RNA with one open reading frame coding for polyprotein. The polyprotein is processed to produce three structural proteins, i.e., capsid (C), precursor membrane (prM), and envelope (E) proteins, and seven non-structural proteins, designated C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 [₁]. The envelope glycoprotein is the major virion antigen responsible for virus neutralization by specific antibodies and several important antigenic properties; it encodes the important biological functions for attachment to cellular receptor, membrane fusion and elicitation of virus neutralizing antibodies, and at least seven nonstructural proteins. The function of envelope protein is to control the receptor binding and fusion with the host cell (₂). The dimeric capsid or core protein represents the least conserved structural protein. Its main function is packaging of viral RNA, forming the nucleocapsid, binding of viral RNA, mediating viral assembly, regulation of the viral lifecycle (₃) together with E, C and the viral genome (₄). The premembrane protein functions as the chaperone for folding and assembly of the E protein. The E protein is responsible for viral attachment to the putative cell surface receptors, fusion with the endosomal membranes upon entry, and mediating protective immune responses in the infected host (₅). The DENV-1 to DENV-4 serotypes constitutes the dengue virus complex within the Flavivirus genus of the family Flaviviridae (₆). A study between antigenically distinct variants of dengue virus in phylogenetic relationship showed that DENV-1 was more homologous to DENV-3 as compared with other serotypes was done.

The in silico prediction of protein subcellular localization had been extensively studied in prokaryotes and eukaryotes but it was not done in the case of viruses; whose proteins were frequently involved during interactions at various subcellular localization, to better recognize the role of proteins in host cells and thereby using it in designing the improved therapeutic infectious disease interventions (₇). The complete proteome of dengue virus functional region was studied because it plays a vital role in the regulation of gene. Bioinformatics tools were used to analyze and identify motifs from the whole proteins of dengue virus serotypes. Motifs are typically 6-30 amino acids and correspond to its active site, substrate or ligand binding site and structurally important segment of proteins.

Tyrosine kinase is an enzyme that transfers a phosphate group from ATP to a tyrosine residue in a protein. It was a subgroup of large class of protein kinases. Phosphorylation of proteins by kinases is an important mechanism in signal transduction for regulation of enzyme activity. The Casein kinase 2 is a serine/threonine-selective protein kinase that is a tetramer of two alpha subunits and two beta subunits. The alpha subunits have catalytic kinase domain. Casein kinase 2 has been implicated in cell cycle control, DNA repair, regulation of the circadian rhythm and other cellular processes (₈, ₉). Protein kinase C is a family of protein kinases consisting of ~10 isozymes. PKCs are activated through the same signal transduction pathway as phospholipase C. It is a family of related serine/threonine kinases and plays a key role in cellular responses such as neurotransmission, gene expression, and cell growth and differentiation (₁₀). Myristoylation is an irreversible, post-translational protein modification found in animals, plants, fungi and viruses. It occurs post-translationally also when previously internal glycine residues become exposed by caspase cleavage during apoptosis (₁₁). Important motif amidation is a required post-translational modification for the bioactivation of many neuropeptides, is catalyzed by a bifunctional enzyme, peptidyl glycine- amidating monooxygenase, in a two-step reaction (₁₂). RGD motifs also contribute in the host cell attachment. The present study was aimed to in silico analyze the whole proteomes of dengue virus variants.

Material And Methods

Collection of sequences

Prediction and identification of motifs

Results And Discussion

The Dengue virus polyprotein, consists of three structural subunit proteins viz. envelope, capsid and premembrane were analyzed theoretically. The molecular weight and isoelectric point of all these proteins were deduced (Table 1).

Figure 1

Table 1: Physico-chemical properties and subcellular localization of whole proteomes of Dengue virus variants

The pI value of envelope protein ranges from 6.83 to 7.52, capsid protein ranges from 12.29 to 12.81 and the premembrane protein ranges from 5.59 to 6.97. It indicates protein stability in a particular isoelectric point (pI). The online Virus-Ploc server was used to predict virus protein localization within virus or targeting the host. The whole proteomes of dengue virus serotypes and their specific location in human cellular localization mechanisms by viral proteins and appropriate subcellular localization predictors were used to predict viral protein localization within the host cell (Table1). Motifs are typically 6-30 amino acids and correspond to the active site, substrate or ligand-binding site and they are structurally important segment of proteins. Several important motifs were identified in the study viz. like amidation, protein kinase C, Tyrosine kinase phosphorylation site, casein kinase 2, N- Glycosylation, myristoylation, cAMP/cGMP binding site, ATP/GTP binding loop A, glycosaminoglycan attachment site and GTP binding elongation factor. All these motifs might play an important role in the function of proteomes of dengue virus. cAMP is a second messenger used for intracellular signal transduction, transferring the effects of hormones like glycogen and adrenaline, which cannot get through the cell membrane. Its main purpose is the activation of protein kinases, also use to regulate the passage of Ca2+ through ion channels (3). In our study, envelop protein have five common motifs like protein kinase C, casein kinase 2, N- Glycosylation, myristoylation, camp / cGMP binding site in all different variant of dengue. However, in DEN 4 three additional motifs GTP-Binding Elongation Factor Signature, Glycosaminoglycan attachment site and Tyrosine kinase phosphorylation site were also identified (Table 2-5).

Figure 2

Table 2: Motifs in Envelope protein of DENV I

Figure 3

Table 3: Motifs in Envelope protein of DENV II

Figure 4

Table 4: Motifs in Envelope protein of DENV III

Figure 5

Table 5: Motifs in Envelope protein of DENV IV

In the capsid protein of dengue virus three motifs were common i.e. protein kinase C, myristoylation and cAMP/cGMP binding site. Whereas, in DEN I and DEN IV Glycosaminoglycan attachment site was identified and in DEN IV amidation has an additional motif was identified. It may facilitate in processing the post transcriptional modification of polyprotein. (Table 6, 7, 8, 9).

Figure 6

Table 6: Motifs in Capsid protein of DENV I

Figure 7

Table 7: Motifs in Capsid protein of DENV II

Figure 8

Table 8: Motifs in Capsid protein of DENV III

Figure 9

Table 9: Motifs in Capsid protein of DENV IV

In the premembrane protein of dengue virus four motifs were common N-Glycosylation, N- Myristoylation, Casein kinase 2 and protein kinase C phosphorylation. ATP/GTP binding site motif A (P-loop) was identified only in DENV III and Glycosaminoglycan attachment site was also identified in DENV IV (Table 10, 11, 12, ₁₃).

Figure 10

Table10: Motifs in Premembrane protein of DENV I

Figure 11

Table11: Motifs in Premembrane protein of DENV II

Figure 13

Table13: Motifs in Premembrane protein of DENV IV

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It has been reported that RNA helicase motif in dengue type 2 the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity [₁₃].

N-glycosylation site, N- myristoylation site, protein kinase C phosphrylation site, casein kinase 2 phosphorylation site, cAMP and cGMP dependent protein kinase phosphorylation site, amidation, cell attachment sequences (RGD), tyrosine kinase, and prokaryotic membrane lipoprotein lipid attachment site motifs was deduced from the sub cellular localization of structural proteins in avian influenza (₁₄). In silico study was done to find the disordered regions in hypothetical proteins of human genome to prove the relationships with disease and drug target selections, biochemical validations, drug target determination of novel folds of human proteome (₁₅).

The present study was carried out to find important motifs which contribute in gene regulation, physiological activity and evolutionary stability of dengue virus. The conserved amino acid residue present in these motifs may be used as a target and design of the potent antiviral drugs / vaccine candidates.

Acknowledgement

Correspondence to

Bioinformatics Centre, Biotech Park, Sector G, Jankipuram Lucknow-226021, U.P, India Email: psomvanshi@gmail.com