Fig 2: Vpr binds both the hHR23A and hHR23B isoforms and the hHR23/XPC DNA recognition complex.a Recombinant GST-hHR23A, residues 1–363, 1–217 and 218–363 and GST-hHR23B, residues 1–409, 1–261 and 262–409 were used in GST-pull down experiments with recombinant VprFL. Three replicates were performed for each experiment yielding similar results. b Vpr mutant binding to hHR23A (top) and hHR23B (bottom). FLAG-tagged hHR23AFL (top) or hHR23BFL (bottom) complexes were immunoprecipitated, via FLAG-tag, from HEK293T cell extracts transiently co-expressing the respective hHR23 isoform and HA-tagged VprFL wild type (WT) or with the following mutations: D52R/W54R (52/54), F69A (69), R80A (80). Cell extracts and hHR23 immune complexes were analyzed by immunoblotting with antibodies specific for the FLAG and HA epitope tags, to detect the ectopically expressed FLAG-hHR23A, FLAG-hHR23B and HA-Vpr proteins. c Vpr F69A mutant binding to hHR23A. Expression levels of the HA-Vpr (F69A) and Vpr WT were varied by transfecting different amounts of plasmids expressing these proteins and their binding to FLAG-hHR23A was assessed as described above. d Vpr binds the hHR23/XPC complex. FLAG-tagged XPC, HA-tagged hHR23AFL and myc-tagged VprFL were expressed alone or in combinations in HEK293 T cells. Cell extracts and XPC immune complexes were analyzed by immunoblotting as described above except that the endogenously expressed hHR23B(en) protein was detected with an antibody specific for the hHR23B isoform. Immune complexes were revealed with fluorescent secondary antibodies and fluorescent signals quantified using a Li-Cor Odyssey imager. Relative levels of the indicated proteins in cell extracts and immune-complexes are shown. b–d Two biological replicates were performed for each experiment yielding similar results. The biological replicate of the experiment shown in panel d is shown in Supplementary Fig. 4. a–d Source data are provided as a Source Data file.

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).