Preliminary Studies On The IBDV-binding Domain Of Hsp90α And Its Involvement In IBDV Infection Of Chicken Bursal Cells

Infectious bursal disease (IBD) is a highly contagious disease in chickens caused by infectious bursal disease virus (IBDV). The disease is characterized by severe immuno suppression and high mortality in young chickens, which causes heavy economical losses to poultry industry worldwide. Receptor-binding is the first step in virus infections and thus critical to the understanding of virus-host cell interactions. It has been shown that that chicken heat shock protein 90a (Hsp90a) is the component of receptor complex for IBDV infection of chicken fibroblast, but its involvement in virulent IBDV infection of chicken bursal cells remains to be defined.Elastin-like polypeptides (ELPs) are engineered pentopeptides with temperature-sensitive reverse phase transition prpperty, which are soluble in the solutions below their transition temperatures and become aggregates above their transition temperatures. Therefore, ELP fusaion proteins can be purified by simple methods such as centrifugation. By combining the phase transition of ELPs and the specificity of virus-receptor binding, a virus receptor-binding capture method has been established in our research group. By using the same strategy, in this preliminary study we investigated the involvement of Hsp90a in virulent IBDV infection of bursal B lymphocytes and its IBDV-binding domain.To define the IBDV-binding region of Hsp90a, the full-length cDNA and the coding sequence for N-terminal 284 amino acids or C-terminal 444 amino acids were amplified from heat-shocked fibroblast DF-1 cells by RT-PCR. Each coding sequence was inserted into prokaryotic expression vector in frame with ELP-coding sequence and transformed into E. coli strain BLR (DE3) for expression. The expression of fusion proteins was induced with IPTG and purified from the cell extract by ELP-mediated inverse transition cycling. The purified fusion proteins were incubated with IBDV and their viral binding was detected by RT-PCR. SDS-PAGE showed that all of the three fusion proteins, namely ELP-Hsp90a, ELP-N284 and ELP-C444, were expressed as soluble proteins, and purified to singular protein bands after one roud of transition cycling. RT-PCR analysis showed that fusion proteins ELP-Hsp90a and ELP-C444, but not ELP-N284 fusion protein, could bined to IBDV. This data indicates that the IBDV-binding region was located in the C-terminal 444 amino acid region of Hsp90a.To investigate the involvement of Hsp90a in virulent IBDV infection of bursal B lymphocytes, the total RNA was extracted from primary bursal cells, bursal B cell line DT40 and fibroblast cell line DF-1 and the Hsp90a transcription was detected by RT-PCR. IBDV and bursal cells were incubated with purified ELP-Hsp90α fusion protein and Hsp90a-sepecific antibody, respectively, and their blocking effects were detected by immunofluorescent and viral titration assays. The results showed that, like DF-1 cells, both primary bursal cells and DT40 cell line were positive for Hsp90α expression. Like the antibody against chicken surface IgM (sIgM), both recombinant Hsp90a and its antibody had partial blocking effects on IBDV infection of bursal cells. These results suggest that the sIgM, Hsp90a was involved in but not the only one receptor for IBDV infection of bursal B cells.