A designed peptide disrupting viral protease cleavage restores cGAS-DNA phase separation and type I interferon responses.

Seneca Valley Virus (SVV) 3C protease is essential for viral polyprotein processing and virion assembly. Meanwhile, it has evolved to cleave and antagonize the multiple innate immune proteins, enabling viral immune evasion. Inhibitors of 3C protease are therefore powerful antiviral agents. Among these, antiviral peptide inhibitors hold particular promise because of their high specificity, strong efficacy, and broad-spectrum activity, and minimal side effects. Here, we developed a dimerization-dependent red fluorescent protein (ddRFP) biosensor system to screen anti-SVV 3C peptides and identified a substrate-competitive decapeptide (P5) that markedly suppresses 3C protease activity. P5 inhibited 3C-mediated cleavage of multiple key immune proteins, including porcine cGAS (pcGAS), porcine Gasdermin A (pGSDMA) and porcine Pro-IL-1β (sPro-IL-1β). Mechanistically, P5 directly interacted with the catalytic His48 site of 3C protease through hydrogen bonding. 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. Our findings establish P5 as a highly promising peptide inhibitor of SVV 3C protease with strong translational potential.