FIG 6.

CSFV NS5A interacts with GBP1. (A to D) CSFV NS5A interacts with GBP1. HEK293T cells were cotransfected with pFlag-GBP1 and either pMyc-NS5A, pMyc-NS5B, or pCMV-Myc (pMyc-EV). The cell lysate was harvested. (A and B) Co-IP was performed using an anti-Flag MAb (1:1,000). The precipitated proteins were analyzed by Western blotting (WB) using antibodies against the Myc and Flag tags (A) and the HA and Flag tags (B). (C) For the GST pulldown assay, GST and the GST-GBP1 fusion protein expressed in E. coli BL21 were purified with glutathione resin. The resin was incubated with Myc-NS5A. The bound proteins were determined by Western blotting using a mouse anti-GST PAb (1:2,000) and an anti-Myc MAb (1:1,000). (D) For the endogenous co-IP assay, PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to co-IP using an anti-GBP1 MAb (1:1,000). (E) The NS5A-GBP1 interaction is independent of RNA. HEK293T cells were cotransfected with pFlag-GBP1 and pMyc-NS5A. The cell lysate was collected and treated with RNase A. Co-IP was performed using an anti-Flag MAb (1:1,000). (F and G) Colocalization of GBP1 with NS5A. (F) Expression plasmids pFlag-GBP1 and pMyc-NS5A were cotransfected into BHK-21 cells and subjected to a confocal assay. (G) PK-15 cells were pretreated with IFN-β, infected with CSFV strain Shimen, and subjected to a confocal assay. The distribution and colocalization of GBP1 and NS5A were examined using a Leica SP2 confocal system.