Intraviral Protein interactions of Chandipura Virus

Abstract

Chandipura virus (CHPV), a member of family Rhabdoviridae and genus Vesiculovirus has recently emerged as a significant human pathogen, causing severe encephalitic outbreaks in different parts of India. Continual outbreaks of CHPV together with the limited scientific knowledge pertaining to protein functions and their associations necessitates the need to generate functional molecular reagents and elucidate the role of viral proteins in its replication and mode of pathogenesis. As an important initial step, protocols were optimized for expression and solubilisation of N (Nucleocapsid), P (Phosphoprotein), M (Matrix) and G (Glycoprotein) proteins with three distinct tags (GST, His and Strep) and the soluble proteins were used for interaction analysis. Purification of CHPV M and G proteins has been reported for the first time using prokaryotic expression system. In order to reveal the intraviral protein-protein interactions of CHPV, comprehensive yeast two-hybrid (Y2H) analysis was carried out involving four proteins of CHPV. All the interactions identified by Y2H were further checked independently by GST pull down and ELISA methods. A total of eight interactions (NN, NP, NM, NG, PP, MM, MG and GG) were identified among four viral proteins. Five of these interactions (NM, NG, MM, MG and GG) are being reported for the first time for CHPV. The key observation that the nucleocapsid protein binds to all four viral proteins i.e., N, P, M and G formed the basis of further experimental work. In order to investigate the interacting regions of N protein involved in these interactions, three regions (N1, N2 and N3) were mapped and the involvement of these regions in the interactions of N protein with N, P, M and G proteins was predicted using computational tools (ZDOCK and RDOCK) and validated by yeast two-hybrid and ELISA based assays.