Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-K K. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, CP P, Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-K K-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-K K-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-K K-mediated responses.z 1999 Federation of European Biochemical Societies.

Two genera, the Respirovirus (Sendai virus (SeV) and human parainfluenza virus (hPIV3) and the Rubulavirus (simian virus (SV) 5, SV41, mumps virus and hPIV2), of the three in the subfamily Paramyxovirinae inhibit interferon (IFN) signalling to circumvent the IFN response. The viral protein responsible for the inhibition is the C protein for respirovirus SeV and the V protein for the rubulaviruses, both of which are multifunctional accessory proteins expressed from the P gene. SeV suppresses IFN-stimulated tyrosine phosphorylation of signal transducers and activators of transcription (STATs) at an early phase of infection and further inhibits the downstream signalling without degrading any of the signalling components in most cell lines. On the contrary, the Rubulavirus V protein targets Stat1 or Stat2 for degradation. Proteasome-mediated degradation appears to be involved in most cases. Studies on the molecular mechanisms by which paramyxoviruses evade the IFN response will offer important information for modulating the JAK-STAT pathway, designing novel antiviral drugs and recombinant live vaccines, and improving paramyxovirus expression vectors for gene therapy.

Background: The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined.

We demonstrate here that Sendai virus (SeV) blocks alpha interferon (IFN-␣) signaling to signal transducers and activators of transcription (STATs) in HeLa cells. IFN-␣-stimulated tyrosine phosphorylation of STATs and subsequent formation of the IFN-stimulated gene factor 3 transcription complex were inhibited in SeV-infected cells, resulting in inefficient induction of IFN-stimulated gene products. None of the components of the signaling pathway-type I IFN receptor subunits Jak1, Tyk2, Stat1, Stat2, and p48-was degraded. Moreover, tyrosine phosphorylation of Jak1 in response to IFN-␣ was unaffected at the early phase of infection, suggesting that oligomerization of the receptor subunits proceeded normally. In contrast to Jak1, IFN-␣-stimulated tyrosine phosphorylation of Tyk2 was partially inhibited. Therefore, this partial inhibition of activation of Tyk2 probably contributes to the subsequent failure in the activation of STATs.

All members of the Paramyxovirinae have retained the open reading frame (ORF) for either or both of the accessory proteins, V and C, within the P gene during evolution (26). This finding had suggested crucial roles of the V and C proteins in a virus life cycle, although their roles had remained an enigma for a long time. Several lines of evidence have accumulated, however, demonstrating that the accessory proteins form a group of antagonists against the host immune system (9, 15-17). The Paramyxovirinae family contains three genera: Rubulavirus, Respirovirus, and Morbillivirus. The V protein of rubulavirus simian virus 5 targets a key factor, signal transducer and activator of transcription 1 (STAT1), on interferon (IFN) signaling for proteasome-mediated degradation, thereby inhibiting IFN signal transduction (1,3,6,7,33,35,48,49). The V proteins of other rubulaviruses, including human parainfluenza virus type 2, mumps virus, and simian virus 41 inhibit IFN signaling likewise by inducing a decrease in the STAT1 or STAT2 level (8,25,30,31,34,35,47,49). In contrast, the respirovirus Sendai virus (SeV), which possesses both V and C ORFs, has evolved functions of the C protein instead of the V protein so as to block IFN signaling (12,18). The SeV C ORF produces a nested set of four C proteins, CЈ, C, Y1, and Y2, which are referred to collectively as the C proteins (5, 14). Translation of CЈ, C, Y1, and Y2 initiates at different positions ( 81 ACG, 114 AUG, 183 AUG, and 201 AUG, respectively) and terminates at the same position (UAA 728 ). In addition to the C protein, the shorter forms, Y1 and Y2, have the ability to inhibit IFN signaling (11,22). The C protein, however, does not lead to degradation of any component on the signaling pathway in most cell types (12,18,24,42,49) except for NIH 3T3 mouse embryo fibroblast (MEF) cells (10, 11). The purpose of the present study was to better understand how the SeV C protein inhibits IFN-␣ signaling without degrading cellular proteins on the signaling pathway.The main pathway of IFN-␣/␤ signaling consists of several components, IFN-␣/␤ receptor subunits (IFNAR1 and IF-NAR2), receptor associated kinases (JAK1 and TYK2), two STATs (STAT1 and STAT2), and IFN regulatory factor 9 (p48) (41). Both STAT1 and STAT2 preassociate with the cytoplasmic tail of IFNAR2 in . Binding of IFN-␣/␤ to IFN receptor leads to aggregation of IFNAR1 and IFNAR2, causing the cross-activation of TYK2 and JAK1 (32). TYK2 then phosphorylates IFNAR1 on Tyr 466 (4), which serves as the docking site for the SH2 domain of STAT2 (45). STAT2 binds to the docking site, followed by the phosphorylation of both STAT2 and STAT1. A current model proposed sequential activation of STAT2 and STAT1 in this order (28). The tyrosine-phosphorylated (pY) STAT2-STAT1 heterodimer then translocates into the nucleus and combines IFN regulatory factor 9 (43) to form IFN-stimulated gene factor 3 (ISGF3) and activates transcription of IFN-stimulated genes (ISGs) by binding to IFN-stimulated response elements (ISREs). Two forms o...