File:41598 2016 Article BFsrep31170 Fig1 HTML.webp

English: Singapore grouper iridovirus (SGIV) protein VP088 expression and localization. (a) Late transcription of orf088 in HX1 cells. Early gene orf086 and late gene orf072 were used for comparison and β-actin was used as internal control. (b) SDS-PAGE, showing expression and purification of recombinant VP088 (rVP88; arrowhead). BL21 bacteria were transformed with vehicle pET32a or VP088-expressing pET88C and induced for expression with IPTG at 0.5 mM and rVP088 was purified for detection. Lysate from BL21 bacteria was loaded as a negative control (blank). (c) Western blot analysis, showing the specificity of αVP088 to detect VP088 expressed in BL21 bacteria and SGIV-infected HX1 cells. (d) Confocal microscopic observation of Immunofluorescence from SGIV-infected HX1 cells, showing expression and subcellular distribution of phalloidin-stained F-actin (red) and αVP088-stained VP088 (green). nu, nucleus; VAS, viral assembly site. Scale bars, 5 μm. (e–j) Localization of VP088 by immunoelectron microscopy. Ultrathin-sectioned cells at 60 hpi with SGIV were stained with αGFP as control or αVP088 to locate the VP088 with immuno-gold. (e) Immunoelectron micrographs of SGIV-infected cells stained with αGFP. (f) Higher magnification of boxed area in (e), showing negative result of immuno-gold labelling. Meanwhile, SGIV capsids at different assembly stages including empty (I), partial assembly (II) and full assembly (III) were detected in VAS. (g–j) Immunoelectron micrographs of SGIV-infected cells stained with αVP088, showing VP088 (immuno-nanogold particles, arrowheads) assembled on the empty capsids (g), partial assembled capsid (h), fully assembled capsids (i) in the VAS and virions outside of VAS (j).