E3 ubiquitin ligase NEDD4 inhibits PEDV infection through ubiquitination and degradation of the viral primase NSP8

Porcine epidemic diarrhea virus (PEDV), a highly lethal alpha coronavirus, primarily infects piglet intestines, yet the role of the intestinal homeostasis regulator NEDD4-a HECT-family E3 ubiquitin ligase-in PEDV infection was previously unknown. This study reveals that NEDD4 inhibits PEDV infection by mediating K63-linked ubiquitination of the viral primase NSP8, targeting a conserved lysine (K170) within an NEDD4-recognized SPP motif. The C2 domain of NEDD4 is critical for this antiviral activity, as deletion of its RDDFLGQVDVPLYPLPT exon abolishes PEDV suppression and disrupts NSP8 binding, with AlphaFold3 structural modeling showing this deletion induces conformational changes at the C2-WW interdomain junction. PEDV infection upregulates NEDD4 expression <i>in vivo</i> and <i>in vitro</i>, and functional experiments confirm that NEDD4 overexpression suppresses viral replication while knockout enhances it. Ubiquitinated NSP8 is degraded via both NDP52-dependent selective autophagy and the ubiquitin-proteasome system, with NEDD4 further amplifying autophagy through beclin-1 upregulation. These findings not only elucidate a host antiviral mechanism whereby NEDD4 restricts PEDV infection through dual degradation of NSP8 but also highlight the therapeutic potential of targeting the NEDD4-NSP8 axis to combat PEDV infection.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a highly lethal alpha coronavirus that causes devastating economic losses in the swine industry worldwide, yet effective control measures remain limited due to an incomplete understanding of host antiviral mechanisms. This study narrows this critical knowledge gap by identifying NEDD4 as a pivotal host defense factor that suppresses PEDV replication through a novel dual-pathway mechanism: mediating K63-linked ubiquitination of the viral primase NSP8 at a conserved motif K170 to trigger its degradation via both selective autophagy and the proteasome system while enhancing antiviral autophagy through beclin-1 upregulation. Our work establishes the NEDD4-NSP8 axis as a promising therapeutic target for the development of much-needed antiviral strategies to combat PEDV and related coronaviral infections.