Investigation of potential cell binding sites on nipah virus attachment glycoprotein

Abstract

Background: Nipah virus (NiV), an emerging zoonotic paramyxovirus, is highly pathogenic with unusual broad host tropism. NiV gains entry into the host cells through a pH-independent membrane fusion event which requires the concerted action of the two membrane glycoproteins, attachment (G) and fusion (F) glycoprotein. This viral entry event is initiated when the G glycoprotein attaches to host cellular receptor, ephrin-B2 or ephrin-B3. The present study aims to map the virus-host interaction, particularly the regions on NiV G that are important for NiV direct binding to cells by using phage display system. Methods & Materials: Five truncated fragments of NiV G extracellular domain were generated by RT-PCR and cloned into phagemidvector, pCANTAB5E. These recombinantphagemidswere transformed into Escherichia coli TG1 for display of truncated NiV G on M13 phage g3p minor coat protein. The binding efficacy of recombinant phages displaying the different regions of NiV G to three NiV susceptible cell lines and the cells protein: African green monkey kidney cells (Vero), human lung fibroblast cells (MRC-5) and human monocytes (THP-1), were evaluated by phage ELISA. Results: A library of recombinant phages displaying truncated NiV G was successfully generated, with a representative titer of at least 1010 cfu/ml. Recombinant phages displaying region of NiV G consisting of amino acids 498-602 (G498-602) demonstrated highest binding to three different cell lines (2.7 x 104 cfu/ml to Vero cells; 6.8 x 103 cfu/ml to MRC-5 cells; 8.7x103 cfu/ml to THP-1 cells) and the binding was dose-dependent. Binding of recombinant phages to Vero cells proteinwas also the highest for G498-602 phages. The region of NiV G from amino acids 71-181 (G71-181), on the other hand, showed highest binding to MRC-5 and THP-1 cells protein. Conclusion: This study demonstrated the first direct binding of NiV G to cells by using phage display system, with findings suggesting that the region of NiV G from amino acids 498-602 plays an important role in NiV direct attachment to cells.