Data that underlies this paper.

Netrin-1, a secreted laminin-related protein, is increasingly recognized for its role in viral pathogenesis, alongside its established functions in neural guidance and immune regulation. We previously identified endothelial lipase (LIPG) as a host factor that facilitates hepatitis B virus (HBV) attachment via heparan sulfate proteoglycans (HSPGs) and/or the sodium taurocholate cotransporting polypeptide (NTCP). Through LIPG-based screening, we identified Netrin-1 as an LIPG-interacting protein, and synthetic peptides derived from Netrin-1 sequences exhibited potent anti-HBV activity. In primary human hepatocytes, Netrin-1 demonstrated antiviral activity against HBV, and in HepG2-NTCP-YFP cells, it inhibited viral attachment and internalization. Mechanistically, Netrin-1 binds to LIPG through heparin-binding motifs in its V and C domains, disrupting LIPG-HBV interactions and displacing LIPG from HSPGs. Furthermore, Netrin-1 binds to the extracellular domain of epidermal growth factor receptor (EGFR), abrogating NTCP-EGFR complex formation and inhibiting EGFR dimerization and phosphorylation, independently of HSPGs. In vivo, recombinant Netrin-1 suppressed the viral infection in humanized hepatocyte chimeric mice. These findings establish Netrin-1 as a multifunctional host factor that interferes with HBV entry, supporting the development of Netrin-1-based therapeutic strategies.