Designed decapeptide P5 inhibits Seneca Valley Virus 3C protease, restoring cGAS-DNA phase separation and type I interferon responses.

The Seneca Valley Virus (SVV) 3C protease is critical for viral replication and has evolved to cleave and antagonize multiple host innate immune proteins, enabling viral immune evasion. This cleavage disrupts essential antiviral pathways, including the cGAS-STING pathway and inflammasome activation, which are crucial for initiating type I interferon responses. Current antiviral strategies often lack specificity or broad-spectrum activity. Inhibitors of 3C protease, particularly peptide-based ones, offer a promising avenue due to their high specificity, strong efficacy, and potentially minimal side effects, addressing a key gap in antiviral therapeutics.

Researchers developed a dimerization-dependent red fluorescent protein (ddRFP) biosensor system to screen for anti-SVV 3C peptides. This led to the identification of P5, a substrate-competitive decapeptide. They then tested P5's ability to inhibit 3C protease activity and its cleavage of key porcine immune proteins: pcGAS, pGSDMA, and sPro-IL-1β. Mechanistic studies investigated P5's interaction with the 3C protease's catalytic His48 site. Further cellular assays evaluated P5's capacity to restore cGAS-DNA liquid-liquid phase separation (LLPS) and enhance downstream antiviral interferon signaling, alongside assessments of cellular permeability, cytotoxicity, and stability.

Decapeptide P5 markedly suppressed SVV 3C protease activity. It effectively inhibited 3C-mediated cleavage of multiple key immune proteins, including pcGAS, pGSDMA, and sPro-IL-1β. Mechanistically, P5 directly interacted with the catalytic His48 site of 3C protease through hydrogen bonding, confirming its substrate-competitive nature. This interaction was crucial for its inhibitory effects.

Remarkably, P5 restored the formation of cGAS-DNA liquid-liquid phase separation (LLPS) by competitively blocking 3C cleavage activity, thereby enhancing cGAS activity and downstream antiviral interferon signaling. Furthermore, P5 demonstrated favorable cellular permeability, low cytotoxicity, good stability, and robust antiviral activity in cellular models, highlighting its therapeutic potential.