The STAT2 activation process is a crucial target of Sendai virus C protein for the blockade of alpha interferon signaling

Abstract

Sendai virus (SeV) C protein functions as an interferon (IFN) antagonist and renders cells unresponsive to both alpha/beta IFN (IFN-alpha/beta) and IFN-gamma. We have recently found the physical association of the C protein with signal transducer and activator of transcription 1 (STAT1) in infected cells. However, involvement of the C-STAT1 interaction in the blockade of IFN signaling has remained unclear. We generated here a series of C mutant proteins that retained or lost the STAT1-binding capacity and examined their effects on IFN-alpha signaling. All of the C mutant proteins with no STAT1-binding capacity lost the ability to inhibit the IFN-alpha response. In contrast, the C mutant proteins retaining the STAT1-binding capacity suppressed IFN-alpha-stimulated tyrosine phosphorylation of both STAT2 and STAT1 to various degrees. Remarkably, their anti-IFN-alpha capacities correlated well with the inhibitory effect on phosphorylation of STAT2 rather than STAT1. In infected cells, the levels of tyrosine-phosphorylated (pY) STAT2 were below the detection level irrespective of duration of IFN-alpha stimulation, whereas the levels of pY-STAT1 strikingly increased after long-term IFN-alpha stimulation. These results suggest that the STAT2 activation process is a crucial target for the blockade of IFN-alpha signaling. An in vitro binding assay with extracts from (STAT1-deficient) U3A and (STAT1-expressing) U3A-ST1 cells suggested the requirement of STAT1 for the C-STAT2 interaction. Furthermore, expression of STAT1 enhanced the inhibitory effect of the C protein on STAT2 activation in U3A cells. The C protein thus appears to participate in the inhibitory process for STAT2 activation through the STAT1 interaction.

FIG. 1.

C mutant proteins expressed as GST fusion proteins and their STAT1-binding capacities. (A) Schematic diagram of constructs of C, Y1, Y2, D1, D2, D3, and C/FS; (B) Western blot analysis of purified GST-C, -Y1, -Y2, -D1, -D2, -D3, and -C/FS. The purified GST-C mutant fusion proteins were subjected to SDS-10% PAGE and detected by Western blot analyses with anti-C antibody. (C) Binding assay with GST-C mutant proteins. HeLa cells were treated with IFN-α for 1 h and then lysed. The total extracts (∼150 μg) were incubated with GST-C mutant fusion proteins (∼2 μg) conjugated to glutathione-Sepharose beads for 1 h. Each of the mixtures was divided by centrifugation into supernatant (sup) and pellet (pt) fractions. The beads were washed three times with the extraction buffer. Proteins in the supernatant and pellet fractions were separated by SDS-7.5% PAGE, transferred to a nitrocellulose membrane, and detected by Western blot analysis with anti-STAT1 (sc-464) or anti-pY-STAT1 (no. 9171) antibody.

FIG. 2.

Establishment of HeLa cell lines, which constitutively express either of C mutant proteins. (A) Extracts (40 μg) from the established cell lines were subjected to SDS-13% PAGE, followed by Western blot analyses with anti-RGS-His antibody. Arrowheads indicate positions of C, Y1, Y2, D1, D2, and D3 proteins. D2 was detected only after long exposure (lane 9). (B) Binding assay with Ni-NTA beads and extracts from the established cell lines. The cells were lysed in the extraction buffer. Proteins in the extracts (ex) and those bound to Ni-NTA beads (pt) were separated by SDS-7.5% PAGE, followed by Western blot analyses with anti-STAT1 (sc-464) antibody.

FIG. 3.

Effects of the C mutant proteins on IFN-α-stimulated gene activation and on ISGF3 formation. (A) The established cell lines were doubly transfected with pISRE-TA-luc and pRL-TK-luc. At 20 h posttransfection, the cells were treated or mock treated with IFN-α (1,000 IU/ml) for 6 h and then lysed. The firefly luciferase activity in the extracts, expressed in relative light units, was normalized to renilla luciferase activity. Data represent the mean values of the normalized luciferase activities from triplicate samples. (B) Confluent monolayers of the established cell lines were treated or mock treated with IFN-α (1,000 IU/ml) for 1 h. The extracts from the cells were subjected to EMSA with a ³²P-labeled ISG15 ISRE probe. (C) Confluent monolayers of the established cell lines were treated with IFN-α (1,000 U/ml) for 0 or 16 h. Extracts from the cells were subjected to SDS-6.5% PAGE, followed by Western blot analyses with anti-STAT1 (sc-464) antibody.

FIG. 4.

Effect of the C mutant proteins on tyrosine phosphorylation of STAT1 in response to short (A)- or long (B)-term stimulation with IFN-α. Confluent monolayers of the established cell lines were treated with IFN-α (1,000 U/ml) for 0 h (A and B), 1 h (A), or 16 h (B). Extracts from the cells were subjected to SDS-6.5% PAGE, followed by Western blot analyses with anti-pY-STAT1 (no. 9171) or anti-STAT1 (sc-464) antibody.

FIG. 5.

Effect of the C mutant proteins on tyrosine phosphorylation of STAT2 in response to short (A)- or long (B)-term stimulation with IFN-α. Confluent monolayers of the established cell lines were treated with IFN-α (1,000 U/ml) for 0 h (A and B), 1 h (A), or 16 h (B). Extracts from the cells were subjected to SDS-6.5% PAGE, followed by Western blot analyses with anti-pY-STAT2 (no. 07-224) or anti-STAT2 (sc-476) antibody.

FIG. 6.

Levels of pY-STAT2 (A) and pY-STAT1 (B) at various intervals after IFN-α stimulation in SeV-infected cells. HeLa cells were mock infected or infected with SeV at an MOI of 10. The media were replaced with media containing IFN-α (1,000 IU/ml) at 2 h p.i. The cells were harvested at the indicated times. Extracts from the cells were subjected to SDS-6.5% PAGE, followed by Western blot analyses with anti-pY-STAT2 (no. 07-224) or anti-STAT2 (sc-476) (A) or anti-pY-STAT1 (no.9171) or anti-STAT1 (sc-346) (B) antibody.

FIG. 7.

STAT1-dependent interaction of the C protein with STAT2. (A) HeLa cells (∼10⁷ cells) were labeled with Redivue l-[³⁵S]methionine (1.8 MBq/ml; 37 TBq/mmol; Amersham Pharmacia Biotech) for 4 h. The radioisotope-labeled cell extract (∼100 μg) was subjected to binding assay with GST, GST-D1, or GST-D3. Bound products were separated by SDS-10% PAGE and visualized with the aid of Amplify (Amersham Pharmacia Biotech) according to the manufacturer's instructions. An arrow indicates the position of STAT1. (B) Cytoplasmic extracts were prepared by lysis of HeLa, U3A, or U3A-ST1 cells with a hypotonic buffer (10 mM HEPES-KOH [pH 7.9], 0.25% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, 1 mM DTT). After centrifugation, NaCl concentration of the clarified lysate was adjusted to 100 mM. Large amounts (∼1 mg) of HeLa, U3A, and U3A-ST1 extracts were subjected to binding assay with GST-D1 or GST. The hypotonic buffer containing 100 mM NaCl was used as a washing buffer. Bound proteins were separated by SDS-6.5% PAGE, followed by Western blot analyses with anti-STAT1 (sc-346) or anti-STAT2 (sc-476) rabbit antibody.

FIG. 8.

STAT1 enhanced the inhibitory effect of the C protein on STAT2 activation. U3A or U3A-ST1 cells were infected or mock infected with SeV at an MOI of 10. At 6 h p.i., the cells were treated with IFN-α (1,000 IU/ml) for 0 or 1 h. Extracts from the cells were subjected to SDS-6.5% PAGE (or SDS-11% PAGE for analysis of the C protein), followed by Western blot analysis with anti-pY-STAT2 (no. 07-224), anti-STAT2 (sc-476), anti-pY-STAT1 (no. 9171), anti-STAT1 (sc-346), or anti-C antibody.

FIG. 9.

Schematic diagram for the proposed molecular mechanism by which SeV blocks IFN-α signaling. Black circles indicate tyrosine residues phosphorylated. STAT2 preassociates with not only the cytoplasmic tail of IFNAR2 but also STAT1 in IFN-untreated cells. In infected cells, the C protein binds to the STAT1-STAT2-IFNAR2 complex. Formation of the C-STAT1-STAT2-IFNAR2 complex results in the complete inhibition of STAT2 activation, as well as the partial inhibition of STAT1 activation. This complex formation may also affect activation of TYK2 and JAK1.