# SVCV phosphoprotein hijacks phase separation to immobilize the IRF3-TBK1 signaling axis and suppress interferon antiviral immunity

## TL;DR

This study shows how a fish virus uses a cellular process called phase separation to block the immune system's ability to produce interferon, a key defense against viruses.

## Contribution

The study reveals a novel viral immune evasion mechanism where SVCV-P hijacks phase separation to immobilize IRF3-TBK1 signaling.

## Key findings

- SVCV-P uses phase separation to form condensates that sequester TBK1 and IRF3, blocking interferon production.
- Disrupting SVCV-P phase separation restores interferon signaling and reduces viral replication in vitro.
- Phase-separation-deficient SVCV-P mutants lose immunosuppressive activity, which can be rescued with LLPS-capable domains.

## Abstract

Spring viremia of carp virus (SVCV), a highly pathogenic rhabdovirus prevalent in fish, causes substantial mortality by evading host antiviral immunity; however, the underlying mechanisms remain incompletely understood. This study reveals a novel immune evasion strategy whereby the SVCV phosphoprotein (SVCV-P) hijacks the TBK1-IRF3 signaling axis via liquid-solid phase transition (LSPT), sequestering interferon regulatory factor 3 (IRF3) and inhibiting interferon (IFN) production. Upon stimulation, IRF3 facilitates TBK1 into functional liquid-liquid phase separation (LLPS) condensates, spatially enhancing IRF3 phosphorylation and downstream IFN responses. IRF3 acts as a scaffold via its DNA-binding domain (DBD) and intrinsically disordered region (IDR), while TBK1 incorporates via its kinase domain (KD), ubiquitin-like domain (ULD), and scaffold dimerization domain (SDD). Conversely, SVCV-P, driven by its IDRs and central domain (CD), undergoes robust LLPS, competitively recruiting TBK1 into SVCV-P-TBK1 condensates. These condensates merge with IRF3-TBK1 droplets, forming SVCV-P-TBK1-IRF3 ternary condensates. These subsequently undergo LSPT, immobilizing IRF3 and preventing its nuclear translocation. In vitro reconstitution and domain-deletion assays confirmed key domain roles in mediating LLPS and LSPT. Disrupting SVCV-P LLPS restored IFN expression and reduced viral replication in vitro. Zebrafish infection models demonstrated SVCV-P-mediated LLPS impaired IFN signaling and increased mortality. Phase-separation-deficient mutants (SVCV-PΔIDR) lost immunosuppressive activity; this defect was rescued by chimeric SVCV-P proteins with heterologous LLPS domains. This study unravels a novel LLPS-dependent mechanism for TBK1-IRF3 signalosome regulation and demonstrates how SVCV hijacks phase separation to remodel host complexes into pathological aggregates, providing a paradigm for viral immune evasion and suggesting new antiviral targets.

Understanding interferon (IFN) signaling regulation and viral evasion is central to host-pathogen interactions. The discovery of liquid-liquid phase separation (LLPS) in cellular activities provides a new perspective for such investigations. Spring viremia of carp virus (SVCV), a severe fish pathogen, has potent IFN evasion capabilities, making it an attractive research model. Here, we demonstrate that LLPS spatially enhances IFN production by concentrating interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) into functional droplets, thereby boosting IRF3 activation. However, the SVCV phosphoprotein (SVCV-P) disrupts this via dual phase-separation mechanisms. First, SVCV-P undergoes LLPS to hijack TBK1 into viral-host condensates, sequestering it from IRF3. Second, these droplets merge with host defense droplets, trapping IRF3 within ternary aggregates. This paralyzes IRF3, blocking its nuclear translocation and IFN production. These findings provide new insights into how viruses exploit phase separation to block innate immune signaling, highlighting LLPS as a promising cross-species antiviral target.