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Background & Aims
Hepatitis C virus (HCV) replication/morphogenesis takes place at the membranous web. Viral genome replication occurs in
replicon complexes on the cytoplasmic face of the ER whereas HCV assembly is located on the surface of lipid droplets (LDs). This raises the question about targeting of
de novo synthesized viral genomes from the
replicon complex to LDs and cellular proteins involved in this process such as the LD-associated protein TIP47, also known as cytoplasmic sorting factor.
Methods
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.
Results
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.
Conclusions
TIP47 plays an essential role in the HCV life cycle.
