Pyroptosis mediated by Caspase-1/GSDMD

Pyroptosis mediated by Caspase-1/GSDMD is a crucial antiviral defense. We reveal that PRRSV nucleocapsid protein (N) subverts this pathway by recruiting host ubiquitination machinery. PRRSV N binds Caspase-1 via its N-terminal domain (residues 1-57), driving K48-linked ubiquitination and proteasomal degradation. This suppresses GSDMD cleavage (p30 generation), LDH release, and IL-1β maturation. PRRSV N hijacks deubiquitinase OTUD4 to remove Caspase-1 ubiquitin chains while stabilizing itself through reciprocal feedback. OTUD4 catalytic mutant (C45A) still disrupted Caspase-1 stability, indicating non-enzymatic scaffolding roles. Caspase-1 overexpression inhibited PRRSV replication in 3D4+CD163 and Marc-145 cells, reversed by inhibitor VX765. Thus, PRRSV N exploits OTUD4 to destabilize Caspase-1 and evade pyroptosis. The OTUD4-Caspase-1 axis represents a therapeutic target against PRRSV immunosuppression.由半胱天冬酶-1（Caspase-1）/GSDMD（Gasdermin D）介导的细胞焦亡（Pyroptosis）是一类关键的抗病毒防御机制。本研究揭示，猪繁殖与呼吸综合征病毒（PRRSV）核衣壳蛋白（N）可通过招募宿主泛素化系统（ubiquitination machinery）逃逸该防御通路。PRRSV N蛋白通过其N端结构域（N-terminal domain，1-57位残基）结合Caspase-1，诱导发生K48连接型泛素化（K48-linked ubiquitination）并介导蛋白酶体降解（proteasomal degradation）。该过程会抑制GSDMD裂解（p30片段生成）、乳酸脱氢酶（LDH）释放以及白细胞介素-1β（IL-1β）的成熟。PRRSV N蛋白还劫持去泛素化酶OTUD4（deubiquitinase OTUD4）以去除Caspase-1的泛素链，并通过双向反馈机制稳定自身。OTUD4的催化突变体（C45A）仍可破坏Caspase-1的稳定性，表明其发挥非酶促的支架蛋白功能。在3D4+CD163细胞与Marc-145细胞中，过表达Caspase-1可抑制PRRSV的复制，该效应可被抑制剂VX765逆转。综上，PRRSV N蛋白通过利用OTUD4降低Caspase-1的稳定性，从而逃逸细胞焦亡。OTUD4-Caspase-1轴可作为对抗PRRSV免疫抑制的治疗靶点。