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

Abstract

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

Street RABV induces the cholinergic anti-inflammatory pathway in human monocyte-derived macrophages by binding to nAChr ɑ7, and upregulates the M2-c marker CD163

“…While major immune evasive mechanisms have been identified for the RABV nucleoprotein (N) and phosphoprotein (P), affecting RIG-I activation (Masatani et al, 2013(Masatani et al, , 2011(Masatani et al, , 2010 and the IFN signal transduction pathway (Brzózka et al, 2006(Brzózka et al, , 2005Vidy et al, 2005) or cytokine signaling (Harrison et al, 2020) respectively, related mechanisms for the surface protein RABV-G have not yet been identified. Our results show that exposure of human MDMs to RABV leads to a decreased TNF-ɑ response upon LPS challenge, caused by cytoplasmic retention of NF-κB.…”

Phenotypic Divergence of P Proteins of Australian Bat Lyssavirus Lineages Circulating in Microbats and Flying Foxes

“…Consistent with conservation of essential C-NLS residues (Figure 1A) [14,17], LMB treatment resulted in nuclear accumulation of ABLVi-P and ABLVf-P proteins (Figure 2C,D). However, accumulation of ABLVf-P protein, determined by calculating nuclear to cytoplasmic fluorescence ratio (Fn/c) [13,15,20], significantly exceeded that of ABVLi-P protein (Figure 2C,D). Comparable results were observed in Hela cells (Figure S2).…”

“…Cos-7 cells were transfected for 6 h with plasmid to express viral proteins or controls before treatment with 1000 U/ml IFN for the specified times, followed by IP using the GFP-trap system (Chromotek) according to the manufacturer's instructions; protease inhibitor and PhosSTOP (Roche) were included in lysis and wash buffers [10,13,20]. Proteins were detected by SDS-PAGE and immunoblotting (IB), using anti-pSTAT1 (Invitrogen #33-3400 or Santa Cruz #sc-7988), anti-GFP (Roche #11814460001 or Abcam #Ab6556), and anti--tubulin (Sigma #T8328) antibodies.…”

“…Cells were transfected for 24 h before treatment without or with 5.2 nM Leptomycin B (LMB, a gift from Minoru Yoshida, RIKEN, Japan) for 3 h and imaging live in phenolfree DMEM with heated chamber using a Nikon C1 inverted confocal laser scanning microscope. Images were analysed using Fiji software (NIH) to calculate the nuclear to cytoplasmic fluorescence ratio (Fn/c) corrected for background fluorescence from > 40 cells/group, as described previously [13,26].…”

“…pY-STAT1 translocates to the nucleus and, in association with pY-STAT2 and IRF9, activates transcription of IFN-stimulated genes (ISGs) [9]. The globular C-terminal domain (CTD) of P-protein (Figure 1) binds pY-STAT1 to inhibit signalling [10][11][12][13]. P-protein also undergoes nuclear trafficking via several nuclear localization (NLS) and export (NES) sequences (see Figure 1; [14][15][16][17][18][19]); these were identified in RABV P-protein, but critical residues and trafficking are broadly conserved among lyssaviruses [20].…”