Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production
Figure 1
High-throughput siRNA screen used to identify cellular factors involved in the HCV life cycle.
(A) Schematic of the screen. Primary and validation siRNA screen were performed as described in Supplemental Experimental Procedures. Huh7.5 cells stably expressing Firefly luciferase (FLuc) were infected with the Renilla luciferase HCV reporter virus JcR2a (MOI = 1 TCID50/cell) 72 h after silencing. To determine the impact of knock-down on HCV entry and replication, cells were lysed 72 h after infection and Renilla luciferase activity was measured. To determine the impact of knock-down on virus production, naïve Huh7.5 FLuc cells were infected with supernatants of transfected cells and 72 h later, Renilla luciferase activity was determined. To account for potential cytotoxic effects of siRNAs FLuc was measured in the same lysates by using dual luciferase assay. (B) Approach used to define genes for the extended validation screen. Based on highest z-scores, 108 genes were selected from the primary siRNA screen (grey box). In addition, 105 genes were deduced from a meta-analysis (blue box) by using the indicated data sets (for further details see Experimental Procedures and S1B Table). To each of these data sets a weighting factor (wf) was given, reflecting their reliability and relevance. Nine of the genes selected by this meta-analysis were also identified as hits in the primary screen. Thus, a total 204 genes were used for the validation screen (orange box). (C) Results of the extended validation siRNA screen for entry/replication and assembly/release. The screen was performed in four replicates, using 4 different siRNAs per gene in a 96-well plate format. All replicates were used to compute z-scores for each siRNA (black dots). Hit siRNAs were defined by a z-score of ≤−2 (green line) in case of host dependency factors (HDF, green dots) or a z-score ≥+2 (red line) in case of host restriction factors (HRF, red dots). Only genes for which at least two siRNAs scored positive were considered. We identified 40 genes as HDFs and 16 genes as HRFs. Genes targeted by siRNAs that gave highly significant z-scores are indicated. EIF4A3, eukaryotic translation initiation factor 4A3; EP300, E1A binding protein p300; KCNK6, potassium channel, subfamily K, member 6; CRABP1, cellular retinoic acid binding protein 1; SPCS3, signal peptidase complex subunit 3 homolog; CKAP5, cytoskeleton associated protein 5; ApoE, apolipoprotein E.