Role of cysteines in infectious bronchitis virus envelope protein oligomerization.

Abstract

Coronavirus envelope E protein plays an important role in the virus assembly and morphogenesis. Despite of little sequence homology among the coronavirus E protein family, two or three cysteines located near the C-terminal side of the transmembrane domain are conserved in all E proteins. Infectious bronchitis virus (IBV) has one of the longest coronavirus E proteins with108 amino acid residues and a molecular weight of 12.3 kD. It is also distinctive by having only two conserved cysteines while most other coronavirus E proteins have three. In this study, to facilitate the expression and purification process, IBV E has been fused with a modified β-barrel platform protein. The fusion protein was expressed in E. coli, recovered from inclusion bodies, cleaved by CNBr and IBV E was purified by HPLC. It is the first time to express and purify IBV E and analyze its secondary structure and oligomerization. Gel electrophoresis revealed that IBV E forms mostly monomer in SDS and pentamer in milder detergent PFO. AUC analysis of IBV E in C14 betaine revealed good fitting for monomer-pentamer model. Analysis using ATR-FTIR revealed strikingly high percentage of residues protected from H/D exchange, providing clue for the IBV E membrane topology.