PRRSV NSP2 hijacks host lipophagy via a LIPE-PNPLA2-AMPK-MTOR axis to promote viral replication

Porcine reproductive and respiratory syndrome virus (PRRSV) exploits host lipid metabolism to support its replication by harnessing lipids and their metabolic derivatives. Lipophagy, a selective autophagic process responsible for lipid droplet (LD) degradation and cellular lipid homeostasis regulation, has been implicated in viral infections, however, its specific role in PRRSV replication has not been investigated. In this study, we found that PRRSV infection triggered lipophagy, resulting in LD depletion and elevated intracellular free fatty acids. Mechanistically, the viral protein NSP2 was essential for PRRSV-induced lipophagy by directly interacting with LD-associated lipases LIPE and PNPLA2, facilitating their binding to MAP1LC3/LC3 via LIR motifs. Both interactions were required for lipophagy-dependent viral replication. Furthermore, we demonstrated that the AMPK signaling pathway critically regulated PRRSV-induced lipophagy. AMPK activation promoted viral replication, whereas its inhibition impaired both lipophagy and viral propagation. Conversely, MTOR signaling acted as a negative regulator of lipophagy, with MTOR inhibition promoting this process. Collectively, these findings established that PRRSV hijacked host lipophagy to facilitate viral replication through NSP2-LIPE-PNPLA2 interactions and an AMPK-MTOR signaling pathway. Our work provided mechanistic insights into viral pathogenesis and highlighted potential therapeutic targets for PRRSV prevention and control.Abbreviations: AMPK: AMP-activated protein kinase; co-IP: co-immunoprecipitation; CQ: chloroquine; CMA: chaperone-mediated autophagy; FFA: free fatty acid; HEK-293T: human embryonic kidney 293T; hpi: hour post infection; LAMP1: lysosome associated membrane protein 1; LD: lipid droplet; LIPA/LAL: lipase A, lysosomal acid type; LIPE/HSL: lipase E, hormone sensitive type; LIRs: LC3-interacting regions; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGLL: monoglyceride lipase; MG132: carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NSPs: non-structural proteins; ORFs: open reading frames; PNPLA2/ATGL: patatin-like domain 2, triacylglycerol lipase; PRRSV: porcine reproductive and respiratory syndrome virus; SQSTM1/p62: sequestosome 1.