Abstract
Sendai virus (SeV), which causes respiratory diseases in rodents, possesses the C protein that blocks the signal transduction of interferon (IFN), thereby escaping from host innate immunity. We previously demonstrated by using protein crystallography that two molecules of Y3 (the C-terminal half of the C protein) can bind to the homodimer of the N-terminal domain of STAT1 (STAT1ND), elucidating the mechanism of inhibition of IFN-γ signal transduction. SeV C protein also blocks the signal transduction of IFN-α/β by inhibiting the phosphorylation of STAT1 and STAT2, although the mechanism for the inhibition is unclear. Therefore, we sought to elucidate the mechanism of inhibition of the IFN signal transduction via STAT1 and STAT2. Small angle X-ray scattering analysis indicated that STAT1ND associates with the N-terminal domain of STAT2 (STAT2ND) with the help of a Gly-rich linker. We generated a linker-less recombinant protein possessing a STAT1ND:STAT2ND heterodimeric structure via an artificial disulfide bond. Analytical size-exclusion chromatography and surface plasmon resonance revealed that one molecule of Y3 can associate with a linker-less recombinant protein. We propose that one molecule of C protein associates with the STAT1:STAT2 heterodimer, inducing a conformational change to an antiparallel form, which is easily dephosphorylated. This suggests that association of C protein with the STAT1ND:STAT2ND heterodimer is an important factor to block the IFN-α/β signal transduction.
Highlights
Sendai virus (SeV), which causes respiratory diseases in rodents, possesses the C protein that blocks the signal transduction of interferon (IFN), thereby escaping from host innate immunity
C protein may interact with STAT2 via the association with STAT1, an interaction between C protein and STAT2 was not observed even in the presence of a large amount of STAT1
We previously reported that the association of one molecule of C protein with the STAT1ND homodimer could cause reduction of phosphorylated STAT1 molecules, whereas the association of two molecules of C protein causes reduction of intact STAT1 molecules by complex formation, thereby inhibiting IFN-␥ signaling [33]
Summary
We first confirmed inhibition of signal transduction in cells transiently expressing Y3. 293T cells were transfected with the reporter plasmid pISRE-EGFP together with a plasmid for expression of the FL-C protein and its deletion mutants, FL-Y1 and FL-Y3 (Fig. 1A). Western blot analysis showed that phosphorylation of HA-STAT1 was almost completely inhibited in the presence of FL-C protein (Fig. 2, A and C). Western blot analysis showed that expression levels of wild-type FL-STAT2 and the mutants in U6A cells were comparable (Fig. 6A). FL-C protein was found to inhibit the signal transduction of IFN-␣ in U6A cells expressing FL-STAT2 or its mutants. In the case of U6A-STAT2 and U6A-STAT2R88E cells, ISRE signal transduction was responsive to IFN-␣, and SeV infection moderately decreased EGFP expression, suggesting that SeV weakened the antivirus action of the added IFN-␣ (Fig. 7C). Affinity analysis showed that our experimental data fitted well to a 1:1 binding mode, indicating that one molecule of Y3W125A binds to the disulfide-bonded STAT1ND:STAT2ND heterodimer (Fig. 8E). According to the standard curves to determine concentrations of STAT2NDH85C and Y3, the prepared complex contained almost equimolar concentrations of STAT2NDH85C and Y3 (supplemental Fig. S4C), supporting the results obtained by the SPR experiments
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