Fig 6.

HCV NS5A upregulates IFN signaling molecules in Beclin1-knockdown IHH. (A) Transduction of lenti-NS5A in Beclin1-knockdown cells enhances IFNA1 and IFN-β mRNA expression. Total cellular RNA was extracted from HCV NS5A-transduced siBCN1-IHH or control IHH after 3 days. Intracellular mRNA expression of IFNA1 or IFN-β was measured by quantitative reverse transcription-PCR. GAPDH was used as an internal control, and the fold changes of mRNA are presented after normalization with internal control. The results are presented as means from three independent experiments. (B) Transduction of lenti-NS5A in Beclin1-knockdown cells induces PARP cleavage. Cell lysates were subjected to Western blot analysis with a specific antibody for the detection of PARP. PARP was significantly cleaved to an ∼86-kDa signature peptide in HCV NS5A-transduced Beclin1-knockdown cells compared to control siRNA-treated IHH. The blot was reprobed with an antibody to actin for comparison of equal protein load. (C and D) Autophagy-impaired HCV-infected or NS5A-transduced IHH have increased levels of mitochondrial ROS. IHH treated with control siRNA or BCN1 siRNA were infected with HCV or transduced with lenti-NS5A. The levels of mitochondrion-associated ROS in cells were analyzed by MitoSOX labeling.