Identifying the role of cysteine residues in SARS coronavirus envelope (E) protein oligomerization by using analytical ultracentrifugation and SDS-PAGE.

Abstract

Coronaviruses had caused a pandemic disease, severe acute respiratory syndrome (SARS) around the world in 2003. The viral membrane proteins always become extremely crucial molecules in viral assembly and viral replication. In SARS-Coronavirus, its membrane consists of at least membrane (M), spike (S), and envelope (E) proteins. From those proteins, E protein function is still unknown. Other study has showed palmitoylations of cysteine residues in E protein are crucial for protein stability as well as for viral assembly. Meanwhile, in another study of our lab, it was shown that SARS-CoV E proteins formed homopentameric model when it was run in PFO-PAGE. Thus, in this project, it is suggested that cysteine residues in E protein might form disulfide linkages to improve the stability of that pentameric model. By using analytical ultracentrifugation method and SDS-PAGE, we were trying to find out the role of cysteine residues in single, double, and triple-substitution of E protein mutants. The results showed that disulfide bond was not the primary factor in the pentameric formation of SARS-CoV E protein. Further investigation has showed that E proteins were aggregated before they were analyzed in analytical ultracentrifugation sedimentation equilibrium. Thus, to avoid aggregation, future studies needs to use lower protein concentration or higher detergent concentration.