VPS34-mediated autophagosome-lysosome fusion facilitates classical swine fever virus replication.

Classical swine fever virus (CSFV) is a highly contagious and lethal pathogen that poses a major threat to the global swine industry. Despite its economic impact, no specific antiviral therapies are currently available, underscoring the urgent need to elucidate virus-host interactions for therapeutic innovation. In this study, we screened a glucose metabolism-targeted small-molecule library and identified Vps34-IN-1, a selective inhibitor of phosphatidylinositol 3-kinase class III (VPS34/PIK3C3), as a potent suppressor of CSFV replication in a dose-dependent manner. Time-of-addition experiments demonstrate that Vps34-IN-1 predominantly interferes with the late stage of the viral life cycle. Consistently, siRNA-mediated knockdown of VPS34 significantly impairs viral replication, confirming its role as a critical host dependency factor. Mechanistically, pharmacological inhibition or genetic silencing of VPS34 disrupts CSFV-induced autophagic flux. Notably, the CSFV non-structural protein p7 engages in a specific interaction with UVRAG, a pivotal constituent of the VPS34 complex II, and appears to potentiate VPS34-UVRAG complex assembly, thereby facilitating autophagosome-lysosome fusion. Collectively, these findings uncover an unappreciated role of VPS34 in sustaining CSFV replication and highlight its potential as a viable target for host-oriented antiviral intervention. IMPORTANCE: CSFV remains a major pathogen of global concern, causing severe disease in swine and incurring substantial economic losses in the pig industry. The absence of effective antiviral agents underscores the pressing need for host-targeted therapeutic strategies. In this study, we identified Vps34-IN-1, a selective inhibitor of VPS34, as a potent suppressor of CSFV replication in a dose-dependent manner. Remarkably, Vps34-IN-1 also exhibits potent inhibitory activity against other economically important swine viruses, including BVDV, PRV, and PEDV, demonstrating its potential as a broad-spectrum antiviral agent. Knockdown experiments further validated VPS34 as an essential host factor required for CSFV propagation. Mechanistically, the viral p7 protein engages in a specific interaction with UVRAG, a pivotal constituent of the VPS34 complex II, thereby potentially augmenting VPS34-UVRAG complex assembly and facilitating autophagosome-lysosome fusion. These findings delineate VPS34 as a compelling host-oriented antiviral target and open new therapeutic avenues for the control of CSF and other economically significant swine viral diseases.