GB Virus C/Hepatitis G Virus Envelope Glycoprotein E2: Computational Molecular Features and Immunoinformatics Study

Abstract

Introduction: GB virus C (GBV-C) or hepatitis G virus (HGV) is an enveloped, RNA positive-stranded flavivirus-like particle. E2 envelope protein of GBV-C plays an important role in virus entry into the cytosol, genotyping and as a marker for diagnosing GBV-C infections. Also, there is discussion on relations between E2 protein and gp41 protein of HIV. The purposes of our study are to multi aspect molecular evaluation of GB virus C E2 protein from its characteristics, mutations, structures and antigenicity which would help to new directions for future researches.

Evidence acquisition: Briefly, steps followed here were; retrieving reference sequences of E2 protein, entropy plot evaluation for finding the mutational /conservative regions, analyzing potential Glycosylation, Phosphorylation and Palmitoylation sites, prediction of primary, secondary and tertiary structures, then amino acid distributions and transmembrane topology, prediction of T and B cell epitopes, and finally visualization of epitopes and variations regions in 3D structure.

Results: Based on the entropy plot, 3 hypervariable regions (HVR) observed along E2 protein located in residues 133-135, 256-260 and 279-281. Analyzing primary structure of protein sequence revealed basic nature, instability, and low hydrophilicity of this protein. Transmembrane topology prediction showed that residues 257-270 presented outside, while residues 234- 256 and 271-293 were transmembrane regions. Just one N-glycosylation site, 5 potential phosphorylated peptides and two palmitoylation were found. Secondary structure revealed that this protein has 6 α-helix, 12 β-strand 17 Coil structures. Prediction of T-cell epitopes based on HLA-A*02:01 showed that epitope NH3-LLLDFVFVL-COOH is the best antigen icepitope. Comparative analysis for consensus B-cell epitopes regarding transmembrane topology, based on physico-chemical and machine learning approaches revealed that residue 231- 296 (NH2- EARLVPLILLLLWWWVNQLAVLGLPAVEAAVAGEVFAGPALSWCLGLPVVSMILGLANLVLYFRWL-COOH) is most effective and probable B cell epitope for E2 protein.

Conclusions: The comprehensive analysis of a protein with important roles has never been easy, and in case of E2 envelope glycoprotein of HGV, there is no much data on its molecular and immunological features, clinical significance and its pathogenic potential in hepatitis or any other GBV-C related diseases. So, results of the present study may explain some structural, physiological and immunological functions of this protein in GBV-C, as well as designing new diagnostic kits and besides, help to better understandingE2 protein characteristic and other members of Flavivirus family, especially HCV.

Keywords: GB virus C; Immunoinformatics; glycoprotein E2, GB virus C.

Figure 1.. Variation along E2 Protein Sequences of Hepatitis G Virus of GB Virus C Shown by Entropy Plot

Regions above threshold 1 were supposed as high variable regions, and arrows represent these positions. Circle shows border line variation that did not include in variation analysis. Entropy Values (Hx) are a measure of variation at each amino acid position in the set of aligned sequences.

Figure 2.. Amino acid Distribution and Composition

Frequency of each amino acid, rate of hydrophobic/Hydrophilic residues and positive and negative R-Group in E2 Protein are depicted in figure. This Figure 2 shows that hydrophobic residues are significantly more frequent, as it reflects the hydrophobicity nature of most parts of E2 protein.

Figure 3.. Transmembrane Topology of E2 Protein

Red color: Transmembrane region of E2 protein, Blue color: Inside regions and violet color: Outside membrane regions. Vertical axis and horizontal axis are probability of prediction (transmembrane, inside or outside) and order of amino acids in protein sequence of E2 protein, respectively.

Figure 4.. Representation of Predicted Glycosylation and Phosphorylation Sites

A shows glycosylation, and B represents phosphorylation. Details of each plot are arranged in Table 2.

Figure 5.. GOR IV Secondary Structure Prediction Method

Graphic visualizes the prediction. Blue; Alpha Helix (α-helix), Red; Extended Strand (β-strand), Violet; Other states (Coils).

Figure 6.. Selected Profiles Were Hydro, Flexi, Access, Turns, Surface, Polar and Antigenic and Respective Thresholds Were 1.9, 2, 1.9, 2.4, 2.3, 1.8 and 1.9. Combination of properties (Comb4).

Figure 7.. Antigenicity Prediction Plot of E2 Protein by Using Kolaskar-Tongaonkar Algorithm

Regions with antigenic propensity scale upper 1 are antigenic regions. Threshold, average, maximum and minimum antigenicity were 1.000, 1.058, 1.259, and 0.866 respectively. Window size and center position were 7 and 4, respectively.

Figure 8.. Predicted 3D Structure of the E2 Protein and Visualization of Epitopes and Variations Regions

Epitopes predicted by different methods and outer membrane region were shown by spheres representation in 3D Structure. Hyper Variable Regions (HVR) represent by surface representation.

Figure 9.. Ramachandran Plot of Predicted Model for the E2 Protein of Hepatitis G Virus

RAMPAGE Server Considers Torsional Angles ψ Against φ of Amino Acid Residues in Protein Structure and Results Defining in Favored, Allowed and Outlier Categories.