Structural Basis of the Inhibition of STAT1 Activity by Sendai Virus C Protein.

UNLABELLED: Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/ß) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr(701) by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/ß. IMPORTANCE: Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/ß) and IFN-γ by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr(701) residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr(701), further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-γ. We also discuss the possible mechanism of inhibition of the IFN-α/ß pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

Structural modeling and analysis of dengue-mediated inhibition of interferon signaling pathway.

Dengue virus (DENV) belongs to the family Flaviviridae and can cause major health problems worldwide, including dengue fever and dengue shock syndrome. DENV replicon in human cells inhibits interferon α and ß with the help of its non-structural proteins. Non-structural protein 5 (NS5) of DENV is responsible for the proteasome-mediated degradation of signal transducer and activator of transcription (STAT) 2 protein, which has been implicated in the development of resistance against interferon-mediated antiviral effect. This degradation of STAT2 primarily occurs with the help of E3 ubiquitin ligases. Seven in absentia homologue (SIAH) 2 is a host protein that can mediate the ubiquitination of proteins and is known for its interaction with NS5. In this study, comprehensive computational analysis was performed to characterize the protein-protein interactions between NS5, SIAH2, and STAT2 to gain insight into the residues and sites of interaction between these proteins. The objective of the study was to structurally characterize the NS5-STAT2, SIAH2-STAT2, and NS5-SIAH2 interactions along with the determination of the possible reaction pattern for the degradation of STAT2. Docking and physicochemical studies indicated that DENV NS5 may first interact with the host SIAH2, which can then proceed towards binding with STAT2 from the side of SIAH2. These implications are reported for the first time and require validation by wet-lab studies.