Fig 1. Protein-protein interaction between hA3G and HCV NS3.
(A) NS3 protein in immunoprecipitates was detected with anti-HA antibody (hA3G-HA) by western blot. Supernatants from cell lysates of HCV-infected Huh7.5 cells transfected with phA3G-HA expression vector (lane 4) or plasmid control (lane 3) were incubated, in turn, with HA-specific antibody and Protein A Agarose; the agarose was washed with DPBS, then the immunoprecipitates were boiled and analyzed with western blot using anti-HA or anti-NS3 as a probe. Lane 1, hA3G-HA positive control; lane 2, NS3 positive control. (B) hA3G was bound to maltose-binding protein (MBP)-NS3/4A in vitro. MBP-NS3/4A was firstly bound to amylose resin column, followed by interaction with cell lysates of the Huh7.5 cells transfected with hA3G-HA expression vector or plasmid control; after eluted with eluting buffer, the elutriants were analyzed with western blot probed with anti-NS3 or anti-HA antibody. Lane 1, MBP-NS3/4A positive control; lane 2, hA3G-HA positive control; lane 3, negative control (elutes of lysates of Huh7.5 cells transfected with control plasmid); lane 4, negative control (elutes of free-cells lysates); lane 5, negative control (elutes of lysates of Huh7.5 cells transfected with plasmid hA3G-HA,using amylose resin column with no pre-binding of MBP-NS3/4A); lane 6, sample (elutes of lysates of Huh7.5 cells transfected with plasmid hA3G-HA). (C) Intracellular distribution of hA3G (red signal), HCV core (green signal, left) as well as HCV NS3 (green signal, right) in HCV-infected Huh7.5 cells was analyzed with immune fluorescent staining; blue signal: nucleus. The histograms show the fluorescence intensity analysis results from ROI 1,ROI 2 and ROI 3 in the merged panel using Volocity Demo software. Correlation coefficients for each couple of intensity values (RCore/hA3G or RNS3/hA3G) are shown. The average correlation coefficient is 0.084 ± 0.032 for hA3G and HCV core (RCore/hA3G) and 0.558 ± 0.031 for hA3G and NS3 (RNS3/hA3G).