Fig 1: Marine medaka HSP90ab1 (MmHSP90ab1) interacts with RGNNV capsid protein (CP).(A) The lysates of hMMES1 cells transfected with pEGFP-N3 (Lane 1) or pEGFP-CP (Lane 2) plasmids. The precipitated cell proteins with affinity to GFP (lane 3) or GFP-CP (lane 4) were separated by 10% SDS-PAGE and stained with Coomassie blue. Lane M, protein marker. Black arrows indicated the specific bands for mass spectrometry. (B) HEK293T cells were cotransfected with pCMV-Flag-MmHSP90ab1 and pCMV-Myc-CP plasmids. MmHSP90ab1 (Green) and CP (Red) were detected by immunofluorescence staining with anti-Flag or anti-Myc abs, respectively. Nucleus were stained by Hoechst 33342, Bar = 10 µm. (C) Lysates from HEK293T cells transfected with pCMV-Flag-MmHSP90ab1 and pEGFP-CP or pEGFP-N3 were subjected to immunoprecipitation with anti-GFP abs. Immunoprecipitates and whole-cell lysate (Input) were immunoblotted with anti-GFP and anti-Flag abs. (D) The lysates of HEK293T cells transfected with indicated plasmids were pulled down with purified His-CP or His proteins. The proteins bound to CP and the input were immunoblotted with anti-His and anti-Flag abs. (E) pCMV-Flag-LjHSP90ab1 or pCMV-Flag-BsHSP90ab1 and pCMV-Myc-CP were cotransfected into HEK293T cells. The cell lysates were immunoprecipitated with anti-Flag abs. The immunoprecipitates and input were immunoblotted with indicated antibodies.
Fig 2: Model of the role of MmHSP90ab1 in RGNNV entry into host cells.During infection, RGNNV might attach to cells by the interaction between CP and HSP90ab1, HSC70 or other receptor(s) on the cell surface. Then, CP, its receptor(s), and clathrin formed a protein complex that facilitated RGNNV internalization through the clathrin-mediated endocytosis pathway.
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