Fig 2: The polymerase activity and replication of influenza virus in 293T cells overexpressing ARNT. (A) The effect of ARNT on the polymerase activity of GS/65. 293T cells were transfected with two plasmids expressing Firefly and Renilla luciferases and the other indicated plasmids. At 36 h p.t., the cell lysates were subjected to the luciferase assay described in the Materials and Methods. The overexpression of ARNT inhibits the replication of GS/65 (B,C). 293T cells were transfected with pCAGGS-ARNT and pCAGGS empty vector, and the overexpression of ARNT in 293T cells was confirmed by Western blotting (B). At 24 h p.t., the cells were infected with GS/65 at an MOI of 0.001. The supernatants were collected at the indicated timepoint. Virus titers in the supernatants were determined in embryonated eggs (C). * p < 0.05; ** p < 0.01.

Fig 3: The effect of ARNT on viral genome replication and transcription. HeLa cells treated with ARNT-specific siRNA (siARNT) and non-targeting siRNA (siScr) were infected with GS/65 virus at an MOI of 2. At 8 h p.i., the RNA was extracted, and then the expression levels of vRNA (A), cRNA (B), and mRNA (C) of the NP gene were determined by using qRT-PCR. Nuclear proteins were extracted for Western blot analysis (D). ** p < 0.01.

Fig 4: Interaction between subdomains or domains of ARNT and PA in 293T cells. (A) Schematic diagram of the full-length ARNT and ARNT mutants tested in this study. (B) Co-immunoprecipitation analysis of the interaction between PA and ARNT mutants. 293T cells were co-transfected with pCAGGS-PA-Flag and different c-Myc-tagged ARNT mutants. At 48 h p.t., cell lysates were immunoprecipitated with an anti-Flag antibody followed by Western blotting with anti-c-Myc and anti-Flag antibodies. (C) Schematic diagram of the full-length PA and PA mutants. (D) Co-immunoprecipitation analysis of the interaction between PA mutants and ARNT. 293T cells were co-transfected with pCAGGS-ARNT-c-Myc and different Flag-tagged PA mutants. At 48 h p.t., cell lysates were immunoprecipitated with an anti-Flag antibody followed by Western blotting analysis with anti-c-Myc and anti-Flag antibodies. *, the target protein.

Fig 5: The interaction between ARNT and PA was analyzed by using co-immunoprecipitation assay. (A) The endogenous expression of ARNT in A549, 293T, and HeLa cells. (B) The interaction between PA and ARNT in 293T cells. 293T cells were co-transfected with the pCAGGS-PA-Flag and pCAGGS-ARNT-c-Myc plasmids. The cell extracts were subjected to co-immunoprecipitation assays by using either anti-Flag or anti-c-Myc antibodies to capture the immune complex. The bound proteins were then analyzed reciprocally by Western blotting with an anti-PA antibody to detect anti-c-Myc immunoprecipitates and an anti-ARNT antibody to detect anti-Flag immunoprecipitates. (C) The interaction between viral PA and endogenous ARNT in HeLa cells. HeLa cells were infected with GS/65 at an MOI of 2. At 24 h p.i., the cell extracts were immunoprecipitated with a rabbit anti-ARNT antibody or anti-control IgG antibody, followed by Western blotting with an anti-ARNT antibody and an anti-PA antibody.