Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus

Wang, Yuan; Li, Yan; Ding, Tianbing

The conventional method for identifying virus receptors is a virus overlay protein-binding assay (VOPBA), where virions or virus attachment proteins (VAPs) are applied (20, 32) . In a VOPBA, cell membrane protein extracts from virus-susceptible cells are first subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with virions or VAPs, and, finally, the specific virus-binding protein is indicated by specific enzyme-conjugated mAbs against the virus. However, VOPBA results cannot distinguish the virus-binding protein but only its size (molecular weight) (33, 34) . For a successful VOPBA experiment, a single polypeptide must exhibit the tentative receptor activity, bind viruses without other membrane components and maintain binding activity in electrophoresis detergents (32) . As it failed in a previous trial of VOPBA on BHK-21 cells in our laboratory, we used a Co-IP assay (35) instead to identify the JEV-binding protein with the advantage of a direct virus-membrane protein interaction under conditions that mimic physiological conditions and more natural conformations to enhance the certainty of protein binding. Due to the possible involvement of third party proteins unrelated to JEV binding in the immunoprecipitates, MS was necessarily introduced to probe the proteins in the precipitates and provide clues to the candidate JEV receptor (JEVR) protein(s) for further verification.