Heat Shock Protein 70 Is Associated with Replicase Complex of Japanese Encephalitis Virus and Positively Regulates Viral Genome Replication
Figure 4
Interaction of Hsp70 with components of the JEV replicase complex.
(A) Interaction of Hsp70 with JEV NS3. 293T cells were co-transfected with Myc-tagged Hsp70 plasmid and Flag-tagged NS3 plasmid or vector. Cell lysates were harvested 36 h after transfection for co-IP experiments with anti-Flag antibody. The precipitates were then analyzed by Western blotting with anti-Flag or anti-Myc antibody. (B) Association of Hsp70 with NS3 and NS5 proteins and viral RNA in JEV-infected cells. 293T cells were transfected with Hsp70-Myc expressing plasmid or vector before being mock-infected or infected with JEV at an MOI of 1.0. Cell lysates were harvested at 36 h p.i. and subjected to immunoprecipitation with antibody against Myc, followed by Western blotting for the detection of NS3 and NS5. The RNA were extracted from the precipitates and then subjected to RT-PCR for detecting viral RNA. RNA extracted from cells infected and uninfected with JEV was used as positive control (PC) and negative control (NC) respectively. (C) Colocalization of Hsp70 with NS3, NS5 and dsRNA in JEV-infected cells. 293T cells were transfected with Hsp70-Myc plasmid, followed by mock-infection or JEV-infection. Cells were fixed at 36 h p.i. and were subjected to indirect immunofluorescence to detect Hsp70 (red) and NS3 (green), NS5 (green) and dsRNA (green) respectively. Nuclei are shown by DAPI (blue) staining. The images of cells were acquired with a confocal laser scanning microscope (Carl Zeiss MicroImaging, Inc.).