Fig 2: NRG1 and subsequent down-regulation of ErbB3 enhances replication of HCV.(A) Huh7.5 cells were pretreated with 25 ng/ml of the NRG1 homologue HRG-1β as indicated and subsequently infected with 1 MOI of the HCVcc strain JC1. Seventy-two hours after infection total RNA was prepared and abundance of the HCV genome was determined by rtPCR. (B) Huh7.5 cells were transfected with control siRNA or ErbB3-specific siRNA as described in Materials and Methods. Forty-eight hours after transfection cells were infected with the HCVcc strain JC1 using 1 MOI. After another 48 hours total RNA was extracted and analysed for the abundance of HCV RNA genomes and of succinate dehydrogenase complex subunit A (SDHA) by rtPCR. Semi-quantitative PCR results were calculated as outlined in the legend to Fig 1 from at least three independent experiments and are presented means + SEM.

Fig 3: HCV induces the expression of the ErbB3 ligands NRG1 and Epiregulin.(A) Total RNA was isolated from Huh9-13 replicon cells or Huh7 control cells. Transcript levels of NRG1, NRG2 and Epiregulin were quantified by rtPCR as outlined in figure legend to Fig 1 and the Material and Methods section using primer pairs described in the supporting information (Table A in S1 File). (B) Supernatants from cell cultures of Huh9-13 cells comprising the subgenomic HCV replicon or Huh7 cells for control were analysed for production and release of soluble NRG1 using ELISA specifically recognizing human NRG1. (C) Huh7.5 cells were infected with the HCVcc strain JC1. Total RNA was prepared 72 hours after infection and NRG1 was determined by rtPCR. For (A) to (C) the results are expressed as fractions of the normalized value of the control which was set to 1; data are presented as mean + SEM of at least three independent experiments.

Fig 4: 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.

Fig 5: HCV mediated upregulation of NRG1 expression involves the transcription factor Sp1.(A) Huh7 or Huh9-13 cells were treated with Mithramycin A at the concentrations indicated or left untreated for control. Four and 16 hours after addition of Mithramycin A, total RNA was isolated and NRG1 mRNA expression was quantified by rtPCR. Values were normalized to those obtained from the control cell line Huh7. (B) Huh9-13 cells were transfected with a Sp1-specific siRNA. 72 hours later total RNA was isolated and abundance of Sp1, NRG1 and ErbB3 mRNA was quantified by rtPCR (left, middle and right subpanel, respectively) as outlined in the figure legend to Fig 1 and the Material and Methods section. Values were normalized to those obtained with control siRNA-transfected cells. (C) Huh7.5 cells were transfected with control siRNA or Sp1-specific siRNA as indicated and 48 hours later cells were infected with the HCV strain JC1 using a MOI of 1 TCID50/cell. After additional 48 hours total RNA extracts were prepared and amounts of Sp1 and NRG1 RNA was determined by rtPCR. (D) Huh7 cells were transfected with a Sp1-encoding plasmid and 48 hours later total RNA was extracted and amounts of Sp1-, NRG1- and ErbB3-specific transcripts were quantified by rtPCR. Semiquantitative PCR results were calculated as outlined in the legend to Fig 1 and data are presented as mean + SEM.