SUMOylation-dependent degradation of nucleocapsid is responsible for Pestivirus uncoating.

Classical swine fever virus (CSFV), a highly virulent member of the Pestivirus genus, is one of the most significant pathogens within this group. Although uncoating is a prerequisite for productive infection, the molecular determinants orchestrating this process remain obscure. The Core protein, functioning as the viral nucleocapsid, plays a pivotal role in the uncoating cascade. This study delineates a SUMOylation-dependent, ubiquitin-independent proteolytic mechanism essential for CSFV uncoating. The valosin-containing protein (VCP/p97) preferentially associates with SUMO1-modified Core, directing it toward degradation via the 26S proteasome, specifically through engagement with PSMB2 and PSMD2 subunits. Site-directed mutagenesis of the SUMOylation motif abolishes VCP-mediated degradation, substantiating its functional indispensability. Fluorescent tracking of CSFV virions using molecular beacon and quantum dot labeling further reveals that VCP governs the endosomal trafficking of viral particles from early to late endosomes-an essential step for capsid disassembly and genome release. Moreover, VCP operates in concert with NPL4 and UFD1, enabling the translocation of SUMOylated Core toward the proteasomal machinery. Collectively, these findings uncover a previously uncharacterized SUMO1-VCP-PSMB2/PSMD2 axis that couples intracellular trafficking with proteasomal disassembly of the CSFV Core, providing mechanistic insights into Pestivirus uncoating and nominating host factors as promising antiviral targets. IMPORTANCE: The fusion of the viral membrane and genome release are hallmark events of enveloped virus infections. However, the related dynamic mechanisms of most viruses remain poorly understood. Here, we demonstrate that VCP directly interacts with the CSFV core protein, and the core protein undergoes proteasomal degradation mediated by the PSMD2 and PSMB2 subunits, with VCP acting as a critical mediator. Surprisingly, this degradation process is independent of ubiquitination but exhibits a strong correlation with the SUMOylation of the nucleocapsid protein. In addition, we found that CSFV genome uncoating occurred in late endosomes, a process regulated by the host VCP. Depletion of VCP prevents viral trafficking to late endosomes and thereby disrupts uncoating efficiency. This is the first evidence implicating SUMOylation in viral uncoating. Deciphering the molecular intricacies governing viral uncoating is pivotal for propelling the development of broad-spectrum antiviral therapeutics aimed at the Pestivirus genus.