Figure 2
HCV NS3 triggered Smurf2 mediated proteasomal degradation of QPRT. (A) Reciprocal co-IP to assess the molecular interaction between the co-expressed NS3-Flag and HA-QPRT in 293T cells using the indicated themes. Mouse IgG was used as a negative control. (B) Interaction between co-transfected QPRT-iRN97 and iRC98-NS3/4 A in HeLa cells were assessed for BiFC signals (iRFP channel) by confocal microscopy. The nucleus was counterstained with DAPI (blue channel). Scale bar, 50 μm. (C) Reciprocal co-IP to assess interactions between the nascent NS3 and endogenous QPRT after Huh7.5.1 cells were infected with HCV for 48 h. Mouse IgG was used as a negative control. (D) Immunoblotting of HA-QPRT cleavage in the presence of co-expressed Flag-NS3/4A in 293T cells for 24 h. Reduction of QPRT (top panel) and lack of cleaved fragments of QPRT (middle panel, long exposure). (E) Ubiquitination of myc-QPRT in the presence of HA-Ubi was assessed by co-IP after Huh7.5.1 cells were transiently co-transfected with Flag-NS3/4A, or infected with HCV, for 24 h. IP efficiency was measured by immunoblotting with Myc antibody, and HCV replication was assessed with NS3 antibody in WCL. (F) Degradation of endogenous QPRT was assessed by immunoblotting after Huh7.5.1 cells were infected with HCV for 48 h, in the absence or presence of MG132 treatment for additional 12 h. (G) Degradation of Myc-QPRT was monitored by immunoblotting with co-transfected E3 ligases, Flag-Smurf2, V5-TRIM25 or Flag-HOIP, in Huh7.5.1 cells for 48 h. (H) co-IP to measure interaction between Myc-QPRT with NS3/4A in the presence of co-transfected mock or different Smurf2 siRNA after HCV infection of Huh7.5.1 cells for 48 h. (I) co-IP analysis of the co-expressed Flag-Smurf2 with HA-NS3 and Myc-QPRT in Huh7.5.1 cells. Mouse IgG was used as a negative control. (J) Decay of Myc-QPRT and different mutants as indicated was compared by immunoblotting with or without co-transfected Flag-Smurf2 in Huh7.5.1 cells. Full-length gels and blots are included in the Figures S6 and S7.
