Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein☆
Introduction
Adenoviridae viruses are nonenveloped, double-stranded DNA viruses with an icosahedral capsid comprising 240 hexons and 12 vertex capsomeres [1], [2]. The human adenovirus (HAdV) can be classified into 6 species (A–F) on the basis of hemagglutination and genomic properties [3], consisting of 51 serotypes defined mainly by neutralization criteria [4], [5]. HAdVs can cause a broad spectrum of human infective diseases [6], [7], [8], [9], among which upper respiratory tract infection and pedo-pneumonia caused by serotypes 3 and 7 are particularly serious [5], [10], [11]. Especially in northern China, the major epidemic strains are HAdV3 and HAdV7 [12], [13]. There is as yet no effective curative antiviral medicine or vaccine with which to treat these diseases.
The major coat protein of HAdV is hexon (i.e., a homotrimer protein comprising three monomers A, B and C) [14]. It has been shown that antibodies stimulated by a hexon can neutralize an HAdV-mediated viral infection, and that this neutralization reaction is type-specific [15]. The tower region of this hexon homotrimer contains a large number of type-specific neutralizing epitopes (B-cell epitopes). Identifying these type-specific neutralizing epitopes is of great significance in several areas of HAdV research, including the molecular design of a HAdV vaccine [16], [17], [18], the development of a rapid HAdV diagnostic agent and preparing an antiadenovirus medicine [19], [20]. But very little is currently known about the mapping of type-specific B-cell neutralizing epitopes of hexons in many serotypes of HAdV. Although it is possible to locate hypervariable regions (HVRs) [14], [21] using the multiple sequence alignment (MSA) method [22], it is difficult to identify the type-specific neutralizing epitopes and to obtain the specific three-dimensional (3D) conformation of the epitope peptides for a specific serotype. The 3D conformation of hexon protein can provide relevant information about epitopes. The hexon structures of HAdV type 2 (HAdV2) and HAdV type 5 (HAdV5) [23], [24] are available in the Protein Data Bank (PDB) [25], but these structures have inherent amino acid deletions and disruption of the peptide chains. In addition, related documents show that these structures are only the conserved core region of the HAdV hexon protein [15], and do not provide information of the complete tower structure, which contains the type-specific neutralizing epitopes.
This study investigated two characteristics of epitopes on HAdVs hexons – namely B-cell neutralizing epitopes and type-specific epitopes – using a combination of molecular simulation technology [26] and bioinformatics evolutionary trace (ET) [27], [28] analysis. The 3D structure of the HAdV type 3 (HAdV3) hexon was determined using molecular simulation/homology modeling [26], [29], [30], and the solvent-accessible surface area (SAS) [31] of the model was calculated. In addition, a modified ET method that we named reverse ET (RET) was employed. This involved the use of MSA, sites homology calculation, and a purpose-designed epitope-screening algorithm that combines the results from the SAS analysis and sites homology calculation. The presence of five candidate epitope segments was predicted and mapped onto the 3D model of the hexon. Finally, the predicted epitope peptides were synthesized. Two serological experiments were performed to prove the correctness of epitopes prediction: (1) enzyme-linked immunosorbent assay (ELISA) was used to detect the affinity of epitope peptides and anti-HAdV3 serum; (2) Neutralization Test (NT) was used to test the neutralizing effect to HAdV3 of antipeptides sera.
Section snippets
HAdV3 and anti-HAdV3 serum
The HAdV used in this study was an isolated strain obtained from clinical throat-swab specimens. In 2003 and 2004, many children in the Harbin area of China contracted fever [13], and a total of 384 throat swabs were taken from them in the Department of Pediatrics, No. 1 Subsidiary Hospital of Harbin Medical University. A strain of HAdV (namely Harbin04B) was successfully isolated in our laboratory, and cell culture, immunology, and morphological, PCR, and sequencing analyses of the hexon gene
Homology modeling of HEX3
The HAdV3 hexon monomer encoded by hexon gene contains 937 amino acids, and results obtained with the web-FASTA tool have shown that the AdC68 hexon in the PDB (code: 2obe) has the highest sequence homology, at 85.6%. However, this high homology is not balanceable, since in the SCR it is more than 95%, whereas in the tower region (residues: 115–310, 400–510) it is only about 66%, and the structural differences are mainly in the tower regions; therefore, our homology modeling focused on the
Discussion
B-cell epitopes on antigens were initially studied by investigating special structures using X-ray crystal diffraction [24]. This method was effective at predicting epitopes, but both X-ray crystal diffraction and nuclear magnetic resonance (NMR) methods require large and expensive equipment [26]. In the 1980s, Hopp and Woods reported that a hydrophilicity parameter could be used to predict B-cell epitopes [50]. Developments in bioinformation technologies for determining solvent accessibility,
Acknowledgments
We thank Zhiwei Yang, Cheng Xing of Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, for helping with the homology modeling and the MM and MD simulations. We also thank Weijun Lu and Changqing Ying of our laboratory for help with ELISAs and NTs experiments.
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Supported by The National Natural Science Foundation of China (No. 30771909), The Doctoral Co-financing Project of Chinese Ministry of Education (No. 20070226007), The Natural Science Foundation Key Project of Heilongjiang China (No. ZJY0701), Science and Technology Project of Heilongjiang Provincial Education Department (No. 11521172).