FIG. 3.

CKB interacts with HCV NS4A. (A) Structures of CKB constructs used in the present study. A full-length wild-type CKB without an epitope tag (CKB) or with an N-terminal HA tag (HA-CKB), HA-CKB with deletions (aa 1 to 357, aa 1 to 296, aa 1 to 247, aa 1 to 184, and aa 297 to 381 and del297-357), CKB mutant at the catalytic site, Cys-283 (CKB-C283S) or CKB-C283S lacking aa 297 to 357 (CKB-C283Sdel297-357) are shown. HA-CKB was coexpressed with FLAG-tagged versions of each NS protein of strain NIHJ1 (B) or with NS4A of strain JFH-1 (C) in 293T cells and immunoprecipitated (IP) with an anti-FLAG antibody. Immunoprecipitates were subjected to immunoblotting (IB) with anti-HA or anti-FLAG antibody. (D) Each CKB deletion mutant was coexpressed with FLAG-NS4A in 293T cells. Immunoprecipitates were analyzed by immunoblotting. Arrow, CKB; arrowhead, immunoglobulin heavy chain. (E) SGR-JFH1 cells were transfected with the expression plasmid for CKB-C283S, CKB-C283Sdel297-357 or empty vector. At 72 h posttransfection, HCV RNA levels and the expression of CKB and CKB-C283S were determined by real-time RT-PCR and immunoblotting with anti-HA antibody, respectively. For HCV RNA quantitation, data are indicated as averages and standard deviations (n = 3). *, P < 0.05 against the empty vector control. (F) Structure of NS4A and NS4A constructs. FLAG-tagged NS4A (aa 1 to 54) or its truncated mutants (aa 1 to 20, aa 21 to 39, or aa 40 to 54) are shown. (G) Each NS4A deletion mutant was coexpressed with HA-CKB and analyzed as described above. (H) FLAG-NS4A was coexpressed with HA-NS3 or HA-NS3 and CKB, followed by immunoprecipitation with anti-FLAG antibody. Immunoprecipitates were analyzed by immunoblotting with anti-HA, anti-FLAG or anti-CKB antibody.