Lipid droplets and small extracellular vesicles interplay in Japanese encephalitis virus non-lytic release.

Lipid droplets (LDs) and small extracellular vesicles (sEVs) are classically known for lipid metabolism and intercellular communication, respectively. Here, we reveal a mechanistic connection between LD dynamics and sEV-mediated non-lytic release of Japanese encephalitis virus (JEV) from neuronal cells. Using Neuro2A, SHSY-5Y, N9 microglia, and primary cortical neurons, we show that JEV is packaged within sEVs (~200 nm) through an ESCRT-independent, neutral sphingomyelinase 2 (nSMase2)/ceramide-dependent pathway. Virions inside sEVs display a higher JEV premature membrane/membrane protein (PrM/M) ratio compared to those released via the conventional secretory pathway. Although containing a higher proportion of premature virions than mature ones, sEV-associated JEV virions gain an evolutionary advantage by evading immune detection and delivering multiple virions to recipient cells, thereby increasing overall infection efficiency. Temporal profiling showed early cytoplasmic LD enrichment (from 6 hpi), followed by a surge in sEV release from 14 hpi, suggesting sequential roles for LDs and sEVs. nSMase2 inhibition decreased sEV-mediated egress without affecting viral replication but increased cytoplasmic LD abundance, consistent with LD underutilization in multivesicular bodies (MVB) biogenesis. Our findings identify LDs as facilitators of MVB formation and nSMase2 as a key driver of sEV-mediated viral exit, revealing parallel yet coordinated pathways in JEV's stealthy egress. IMPORTANCE: Lipid droplets and sEVs are traditionally regarded as regulators of lipid homeostasis and intercellular communication. We propose that this LD-sEV connection represents a key mechanism enabling JEV egress from neuronal cells. Japanese encephalitis virus (JEV) exploits small extracellular vesicles (sEVs) for non-lytic viral release from neuronal cells. sEV-containing JEV (~200 nm) is released via an ESCRT-independent, nSMase2/ceramide-dependent pathway. A higher precursor membrane protein (PrM)-to-membrane protein (M) ratio in MVBs and sEVs suggests packaging of immature JEV via a non-secretory pathway. LDs facilitate MVB formation, facilitating sEV-mediated JEV release. sEV release drives LD utilization for MVB formation; nSMase2 knockdown blocks sEV-mediated egress and causes cytoplasmic LD accumulation. JEV enters neuronal cells, releases its RNA from late endosomes, and replicates in the cytoplasm. Virions are subsequently released either through the conventional secretory pathway or by packaging into multivesicular bodies (MVBs) and secretion within small extracellular vesicles (sEVs). Maturation of JEV requires cleavage of the premature membrane protein (PrM) into the membrane protein (M). Notably, the PrM/M ratio is higher in virions released via the secretory pathway compared to those packaged within sEVs, where a greater proportion of immature virions are enclosed. Lipid droplets (LDs), derived from the endoplasmic reticulum (ER), interact with MVBs and contribute to JEV release inside sEVs. This process is driven by a ceramide-dependent, ESCRT-independent pathway regulated by nSMase2. Together, the model highlights the coordinated role of the LD-sEV axis in mediating JEV non-lytic egress.