Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family
Fig 7
Inhibition of NRG1 and/or Akt activity results in an impaired establishment of infection of DMSO differentiated Huh7.5 cells and a reduced infectivity of conditioned supernatants.
The experimental procedure and timeline is shematically summarized in (A). For (B) to (D) Huh7.5 cells were treated with 2% DMSO for two weeks prior to infection. Thereafter cells were infected with 2 MOI HCV JC1 for 48 hours according to the recently published protocols to establish persistent infection [23]. 6 hours prior to infection 2μg/ml of neutralizing NRG1 antibody or 2 μg/ml of the corresponding IgG isotype control were added to the cells as indicated. Medium was exchanged 48 hours post infection and afterwards every second day. With every exchange, 2μg/ml neutralizing NRG1 antibody or IgG control and/or 2μM Triciribine were replenished. For (B) cells were fixed two weeks after infection with ice-cold methanol and analysed for NS5A expression by immunofluorescence and laser scanning microscopy using antibodies specific for NS5A. To test for production of infectious virus the medium was changed for the last time either six (C) or thirteen (D) days after infection and incubation was continued for additional 24 hours. Thereafter supernatants were collected and transferred to fresh, untreated cells. After an incubation period of an additional 72 hours cells were fixed with ice-cold methanol and submitted to immunofluorescence analysis for NS5A expression as above. Cell nuclei were stained using Hoechst dye. Bar = 50μm. Confocal images in (B) to (D) are representative for the image sets taken to obtain quantitative data as shown in the respective column diagrams. For analysis cell nuclei and infected cells were counted using Fiji software and the percentage of infected cells is depicted as the mean and SEM from at least three independent experiments. p ≤ 0.05 was considered to be significantly different.