Fig 2: RNA interference screening of host factors of the HCV assembly proteome. Host factors identified as HA-E2 primary and accessory interaction partners (Fig. 5B), HAHA-p7, and HA-ApoE binders (Fig. 4) were silenced by RNA interference with a pool of three siRNAs per target. Moreover, SLC4A7, OS9, SDF2L1, and C5orf42, de novo HA-E2 interactors identified with at least one of the mutants, as well as Rad23A and RAD23B were included as interactors of NGLY1, an HA-E2 interactor. Subsequently, these cells were infected with the reporter virus JcR2a, and RNA replication was measured 48 hours later. The z-score of a knock-down was calculated by subtracting the median of the negative controls on the same plate and dividing by the standard deviations of the controls. Mean z-scores were calculated by averaging over knock-downs targeting the same gene. Values x < 0 indicate a reduction, and x > 0, an increase of HCV replication or assembly and release compared to the mean of controls. On the y-axis, the log10 of the P-values of the scores are plotted. (A) Infectious progeny produced at this time point were passed on to naïve cells, and infection efficiency reflecting assembly of new viral progeny in the silenced cells was quantified 48 hours post second round infection. (B) PI4KA and ApoE (green dots) were silenced as controls for host factors influencing RNA replication and assembly, respectively. Hit calling was based on mean score ≥2 and P-value ≥0.05; t-test was applied following the RNAither pipeline (50), n = 4–6. Host factors meeting these inclusion criteria are highlighted as red circles.

Fig 3: Modulation of RAD23A and B expression influences HCV infectious virus production. (A) We transfected Huh-7.5/FLuc cells with one of three different siRNAs per factor or a pool of these siRNAs. Forty-eight hours post transfection (hpt), we infected these cells with the reporter virus JcR2a. We passed the culture fluid of these cells to naïve Huh-7.5/Fluc cells and quantified infection 48 hours later. The heat map shows infection data normalized for cell viability and RNA replication quantified in the dual luciferase assay (n = 3). (B) In uninfected Huh-7.5 cells, we quantified the abundance of RAD23A and RAD23B mRNA by qRT-PCR and (C) Rad23A and Rad23B protein levels by Western blot at 48 hours post siRNA transfection. Western blot from one representative experiment is shown. (D) Huh-7.5/FLuc cells were transduced with an empty lentiviral vector or vectors encoding RAD23A or RAD23B. Forty-eight hpt, we infected the cells with JcR2a, and 48 hours later, we measured cell viability as well as cell entry/RNA replication. Culture supernatant was transferred to naïve Huh-7.5/FLuc cells to measure infectious virus production 48 hours post second round infection. Samples were normalized to infected cells treated with an empty control vector (n = 7). (E) Protein expression was analyzed 48 hpt (representative experiment). (F and G) Focus-forming unit (FFU) assay using the untagged HCV Jc1 strain in Huh7.5 cells. Cells were transfected with siRNA, and after 24 hours, cells were infected with a multiplicity of infection of 0.1 TCID50/cell . (G) Forty-eight hours post infection, cells were lysed, and Western blot was performed (representative experiment). (F) Supernatant of infected cells was titrated on naϊve Huh7.5 cells, and infections were stopped 48 hours later. Virus was fluorescently stained with an anti-NS5A antibody, FFUs were counted manually, and data were normalized to the scr control (n = 4) (mean ± SD, Kruskal-Wallis test, only significant differences are highlighted by an asterisk, *P < 0.05, **P < 0.005).