FIG. 1.

BGLF4 interacts with a splicing variant and authentic form of IRF3. (A) GAL4DBD-BGLF4(1-293) and a two-hybrid isolate, GAL4AD-IRF3′(282-452), from a HeLa cDNA library were retransformed into PJ69-4A yeast cells and grown on nutrient selective plates (lacking Ura and Leu; lacking Ura, Leu, and His; or lacking Ura, Leu, and Ade). Negative controls (NC) are GAL4AD and GAL4DBD-BGLF4(1-293). Positive controls (PC) are the known interaction partners (Nijmegen breakage syndrome 1 [NBS1] and importin-α) (68). (B) Summary of IRF3, alternative splicing variant IRF3′ (GenBank accession number AAH00660), and the two-hybrid isolate truncated IRF3′ (amino acid 282 to 452). Functional domains of IRF3 including the N-terminal DBD, nuclear export signal (NES), proline-rich region (Pro), IAD, response domain (RD), and transactivation domain are indicated (3, 17, 47, 64, 73). (C) 293T cells were transiently transfected with plasmids expressing E1/BGLF4(1-293) and HA-IRF3′(282-452) separately or together for 24 h. The tagged proteins were then immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. GST antibody was used as a control antibody (Ctrl. Ab) for immunoprecipitation. (D) 293T cells were transiently transfected with plasmids expressing E1/BGLF4(1-293) and HA-IRF3(282-427) separately or together for 24 h. The tagged proteins were then immunoprecipitated and immunoblotted with the antibodies indicated. (E) HeLa cells were cotransfected with vector control (V) or plasmids expressing wild-type BGLF4 (K) or kinase-dead mutant KD (K102I) with IRF3-Flag or IRF3(5D)-Flag for 24 h. The tagged proteins were then immunoprecipitated by anti-Flag antibody and immunoblotted with the antibodies indicated. Whole-cell extract (WCE) was also applied as an input control. (F) HeLa cells were cotransfected with vector control (V) or plasmids expressing wild-type BGLF4 (K) or the kinase-dead mutant KD (K102I) with IRF3-Flag or IRF3(5D)-Flag for 24 h. Cell lysates were then immunoprecipitated by anti-BGLF4 antibody and immunoblotted with the antibodies indicated. (G) In vitro-translated [³⁵S]methionine-labeled HA-IRF3(282-427) was examined for its ability to interact with GST, GST-BGLF4, or GST-K102I as described in Materials and Methods. (H) In vitro-translated [³⁵S]methionine-labeled BGLF4 was examined for its ability to interact with GST, GST-IRF3(282-427), or GST-IRF3.