Enterovirus 71 VP1 Activates Calmodulin-Dependent Protein Kinase II and Results in the Rearrangement of Vimentin in Human Astrocyte Cells
Figure 4
Analysis of the role of vimentin aggresomes in EV71 replication.
(A) Detection of EV71 genome, vimentin and ser 82-p vimentin distribution in virus infected cells showing the co-localization of EV71 genome with EV71 VP1 in perinuclear region. U251 cells were infected with EV71 for 24 h. Cells were fixed, permeabilized, and quenched as described for immunofluorescence. Cells were then hybridized with a FITC-conjugated DNA probe complemented with EV71 VP1 gene (EV71 gen) as described in Material and Methods. Cells were then stained with antibodies to EV71 VP1 (EV71), vimentin (Vim) or ser 82-p vimentin (Vim p82) and subjected to immunofluorescence analysis. CM: Cell morphology. Bar: 20 μm. (B) A study of the distribution of newly synthesized viral RNA, vimentin and ser 82-p vimentin. Newly synthesized RNA in EV71 infected cells (+EV71) was labeled with BrUTP as described in Material and Methods. The incorporated BrU was stained by using a mouse anti-BrdU antibody (BrU; green). After washing with PBS, cells were stained with antibodies to EV71, ser 82-p vimentin (Vim p82) or vimentin (Vim). Newly synthesized RNA (red) and vimentin (green) in uninfected control (−EV71) was also showed. Bar: 20 μm. (C) Analysis of the aggresomes in EV71 infected cells by transmission EM. Cells were infected with EV71 and prepared for EM as described in Materials and Methods. Panel a: TEM showing a high electron density region near the perinuclear (white box) in EV71 infected cell. Bar: 20 μm. Panel b: A magnified view of the boxed section in panel a. Bar: 500 nm. Panel c: A magnified view of the boxed section in panel b, showing irregular aggresomes-like structures and mitochondria aggregated. Bar: 300 nm. Panel d: A magnified view of the white boxed section in panel c, showing the intact viral-like particles and the incomplete viral-like particles were found localized on the surface or aggregated very close to the aggresomes and near to the mitochondria. m = mitochondria. Bar: 150 nm. Panel e: A magnified image of the black boxed section in panel c. The image shows viral-like particles (long black arrow) combined with filament structures (black arrow head). Note the accumulation of virus-like particles (white arrow) in the replication site. Bar: 200 nm. Panel f: An enlargement of the black box area in image e. Note that virus-like particles interact with filament-like structures (white arrow) and arranged very regularly (black arrow). Bar: 100 nm. Panel g: Another magnified view of the viral replication site showing filament-like structures and viral-like particles. Bar: 100 nm. Panels h and i shows an enlargement of the white box in panel e and white box in panel g, respectively. Black arrow in panel h shows a virus-like particle attached to filament-like structure. Short white arrow head in panel i shows filament-like structures. Long white arrow shows a viral –like particle. Bar: 30 nm.