FIG 7.

Domain I of NS5A is required for MxB and NS5A interaction. (A) MxB interacts with NS5A protein. MxB-Myc was coexpressed with Flag-tagged NS3, NS4A, or NS5A. Immunoprecipitation was performed to pull down Flag-tagged proteins by use of anti-Flag antibody. The level of coprecipitated MxB-Myc was determined by Western blotting. (B) MxB-Myc interacts with NS5A-Flag. HEK293T cells were transfected with NS5A-Flag and MxB-Myc wild-type or mutant DNA. After 48 h, immunoprecipitation was performed with anti-Myc antibodies to pull down MxB. The presence of NS5A-Flag in the precipitated materials was detected by use of anti-Flag antibodies for Western blotting. (C) Schematic representation of the key domains of NS5A. The position of each domain is illustrated by amino acid numbers. (D) Deletion of domain I in NS5A eliminates its interaction with MxB. NS5A-Flag and its deletion mutants were coexpressed with MxB-Myc in HEK293T cells. NS5A-Flag and its mutant proteins were precipitated with anti-Flag antibody, and the presence of MxB-Myc in the precipitated materials was detected by use of anti-Myc antibodies for Western blotting. (E and F) MxB disrupts the interaction of CypA and either NS5A(DI) (E) or NS5A(DEYN) (F). Immunoprecipitation was performed with anti-HA antibodies to pull down CypA-HA. Levels of coprecipitated NS5A-Flag and its mutants were determined by Western blotting using anti-Flag antibody. Data are representative of at least three independent experiments.