Structural Elucidation of Viral Antagonism of Innate Immunity at the STAT1 Interface

Abstract

Highlights d Elucidation of the interface of full-length STAT1 and a viral interferon antagonist d The viral protein-STAT1 interface involves multiple distinct surfaces d Ablation of the interaction requires multiple mutations at distinct sites d Loss of viral protein-STAT1 interaction attenuates a rabies virus street strain

Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling

“…Such independent targeting of STAT1 and STAT3 was reported for mumps virus (MUV) V-protein, which induces degradation of both proteins [16,40]. Alternatively, RABV P-protein CTD directly targets STAT1 (as confirmed recently using biophysical techniques [8]), but does not specifically contact STAT3 and rather interacts with STAT3 in the context of activated STAT3-STAT1 heterodimers. The latter is intriguing as it could result in selective antagonism of IL-6-activated pY-STAT3-STAT1 heterodimers without impacting on distinct pY-STAT3 complexes such as STAT3 homodimers, potentially resulting in selective gene regulation.…”

“…In common with other mammalian viruses, evasion of the host antiviral interferon (IFN)-mediated immune response is critical to RABV infection [ 5 – 8 ] and is mediated by virus-encoded IFN-antagonist proteins. A major function of many IFN-antagonists, including the principal RABV antagonist phosphoprotein (P-protein), is the targeting of signal transducers and activators of transcription (STATs) 1 and/or 2, the major mediators of signalling by antiviral type I IFN (IFN-α/β) that represents the principal innate antiviral response [ 9 , 10 ].…”

“…This leads to the formation of parallel dimers, which accumulate within the nucleus and bind to specific DNA elements in the promotor regions of large numbers of target genes, producing distinct profiles depending on the cytokine stimulus and specific STATs activated [ 11 ]. RABV P-protein binds directly to STAT1 via the P-protein C-terminal domain (CTD) [ 6 , 8 , 12 ], with efficient interaction in cells requiring activation by cytokines [ 13 ]. Thus, P-protein interacts strongly with tyrosine-phosphorylated (pY) STAT1-STAT2 heterodimers in IFN-α/β-treated cells, and inhibits nuclear accumulation via a nuclear export sequence in the P-protein N-terminus (N-NES), which effects cytoplasmic localisation of the complex [ 5 , 12 – 14 ].…”

“…P-protein similarly binds and inhibits STAT1 homodimers, the major complex in type II IFN (IFN-γ)-activated cells [ 12 , 14 ]. STAT1 interaction and N-NES activity is conserved across the Lyssavirus genus [ 15 ] and highly significant to disease progression in vivo [ 5 , 6 , 8 ].…”

Antagonism of STAT3 signalling by Ebola virus

“…Reports supporting constitutive nuclear trafficking of STAT3 suggest that STAT3 accumulates in the nucleus in response to cytokine due to intra-nuclear interactions/sequestration, such as through induced DNA binding [24]. We therefore considered that VP24 may inhibit STAT3 nuclear accumulation in U3A cells by inhibiting the capacity of STAT3 to bind DNA, similar to the antagonistic mechanism of RABV P-protein for STAT1, where the P-protein binds proximal to or within the STAT1 DNA binding domain [33,34]. To assess DNA binding by STAT3 directly, we performed electrophoretic mobility shift assay (EMSA) analysis of cell lysates using the m67 probe ( Figure 5), which is a high affinity variant of the sis-inducible element from the C-FOS gene, commonly used to analyse STAT3-DNA binding [35][36][37].…”