Viral replication was studied in HuH7.5 cells using the infectious HCV JHF1 culture system. Proteome analysis was performed by 2D gel electrophoresis and mass spectrometry. Expression of target genes was modulated by siRNA or lentiviral transduction. Confocal microscopy was performed for analysis of subcellular compartments. Protein/protein interactions were studied by co-immunoprecipitations, affinity chromatography, and yeast two-hybrid screens.
Proteome based analysis revealed that HCV replicating cells contain less TIP47 compared to control cells. However, expression analyses demonstrated an increased TIP47 expression in HCV replicating cells. TIP47 binds to RNA-loaded NS5A. Mapping of the binding domain revealed that NS5A binds to the N-terminal PAT domain of TIP47. Overexpression of TIP47 increases the amount of released viruses, while silencing of TIP47 decreases the amount of released infectious particles. Complete knockdown of TIP47 expression abolishes virus replication.
TIP47 plays an essential role in the HCV life cycle.