ISGF3 complex formation in SeV-infected cells. HeLa cells were mock-infected (m) (lanes 1 and 2) or infected with SeV at the indicated times and harvested at 2 h after replacement with fresh medium containing 10³ IU of IFN-α per ml (recombinant human IFN-α2a from Takeda Chemical Industries Ltd., Osaka, Japan) (lanes 2 to 7) or no IFN-α (lane 1). Nuclear extracts were prepared according to the “mini-extracts” method (23, 25) and analyzed by EMSA with a ³²P-labeled ISG15 ISRE probe as described previously (10). The arrow indicates the position of ISGF3. SeVpB (30), a temperature-sensitive mutant isolated from a carrier culture of BHK cells persistently infected with SeV strain Nagoya 1-60 (11), was used for all of the experiments in this study. HeLa cells were grown in Eagle's minimum essential medium (MEM) supplemented with 5% bovine serum and 3% tryptose phosphate broth.

Poor induction of ISG products in SeV-infected cells. HeLa cells were mock infected (lanes 1 to 3) or infected with SeV (lanes 4 to 9), and then the media were replaced at 2 hpi with fresh medium containing 10³ IU of IFN-α per ml (lanes 2, 3, 5, and 6) or no IFN-α (lanes 1, 4, 7, 8, and 9). The cells were harvested at 0 (lanes 1, 4, and 7), 8 (lanes 2, 5, and 8), and 24 (lanes 3, 6, and 9) h after IFN-α treatment. Total-cell extracts (20 μg of protein) prepared according to the method of Lee et al. (14) were subjected to Western blot analysis as described previously (9). Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody. The same blotting membrane was stripped and reprobed. The protein concentration was determined as described previously (9).

Effects of SeV infection on IFN-α-stimulated tyrosine phosphorylation of Jak1 and Tyk2 and on expression levels of the type I IFN receptor subunits IFN-αR1 and IFN-αR2. HeLa cells were mock infected (lanes 1 and 2) or infected with SeV (lanes 3 and 4) at 2 h prior to replacement with fresh medium containing 10³ IU of IFN-α per ml (lanes 2 and 4) or no IFN-α (lanes 1 and 3). The cells were harvested at 15 min after IFN-α treatment. The total-cell extracts (1 mg) were immunoprecipitated with anti-Jak1 (no. 06-272) (A) and anti-Tyk2 (no. 06-638) (B) rabbit polyclonal antibodies (Upstate Biotechnology, Inc., Lake Placid, N.Y.) before Western blot analysis with antiphosphotyrosine mouse monoclonal antibody (sc-7020). The same blotting membrane was stripped and reprobed with anti-Jak1 (J24320) (A) and anti-Tyk2 (T20220) (B) mouse monoclonal antibodies (Transduction Laboratories, Lexington, Ky.). To detect the type I IFN receptor subunits, total-cell extracts (50 μg) were subjected to Western blot analysis with anti-IFN-αR (IFN-αR1) (sc-845) and anti-IFN-α/βR (IFN-αR2) (sc-704) rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc.) (C).

Inhibition of IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 in SeV-infected cells. HeLa cells were mock infected (lanes 1 to 3) or infected (lanes 4 to 9) with SeV at 2 h prior to replacement with fresh medium containing 10³ IU of IFN-α per ml (lanes 2, 3, 5, and 6) or no IFN-α (lanes 1, 4, 7, 8, and 9). The cells were harvested at 0 (lanes 1, 4, and 7), 5 (lanes 2, 5, and 8), or 30 (lanes 3, 6, and 9) min after IFN-α treatment. Total-cell extracts (50 μg of protein) were subjected to Western blot analysis with anti-phospho-(Tyr 701)-Stat1 (no. 9171) (A) and anti-phospho-(Tyr 705)-Stat3 (no. 9131) (C) rabbit polyclonal antibodies (New England Biolabs, Inc.). To detect tyrosine-phosphorylated Stat2, total-cell extracts (500 μg) were immunoprecipitated with an anti-Stat2 antibody (sc-476) (B) before Western blot analysis with antiphosphotyrosine mouse monoclonal antibody (sc-7020) (Santa Cruz Biotechnology, Inc.). Each blotting membrane was stripped and reprobed with anti-Stat1 (sc-464) (A) mouse monoclonal antibody or anti-Stat2 (sc-476) (B) or anti-Stat3 (sc-7179) (C) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.).