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

Wang, Yuan; Li, Yan; Ding, Tianbing

Using western blot analysis, we only produced positive results under non-reducing and non-boiled conditions, which is in contrast to the routine reducing method (data not shown). The novel band at approximately 95 kDa from the cell membrane protein extracts was reacted solely with either anti-human HSP90β mAb recognizing the amino acid sequences 1-100 that correspond to the human HSP90β N-terminus (see mAb manufacturer's fact sheet; http://www. abcam.com/hsp90-beta-antibody-e296-ab32568.html) or anti-JEV mAb against E protein at the same position around 90 kDa (Fig. 2A) . These data suggest that the HSP90β-JEV complex formed as it was recognised by two different mAbs against the two respective complex components. In addition, the non-reducing and non-boiled conditions imply that the JEV binding activity of HSP90β may depend on the proper conformation of its monomers, which is sensitive to the detergents used in electrophoresis. This conformational requirement may partially explain why the VOPBA produced good results for the similar single peptide chain HSP70 (24,54), but did not demonstrate why HSP90β, which is broadly expressed in mouse brain cells (55), did not bind JEV on Neuro2A cells (24) . For mouse microglial (BV-2) cells, HSP family members, including HSP70, HSP90 and GRP78, are also involved in JEV entry, but play a minor role in virus internalization, which is in contrast to CD4 with a major entry role (25) . Furthermore, the confocal laser scanning microscopic examination demonstrated that HSP90β and JEV co-localised along the Vero cell surface (Fig. 2B) , though HSP90β is generally defined as a cytosolic protein.