Decoding extracellular vesicles-mediated encapsulation of enveloped and nonenveloped gut viruses through phosphatidylserine affinity profiling

Abstract

Viruses are generally classified as enveloped viruses (EnVs) or nonenveloped viruses (non-EnVs), based on the presence of a lipid membrane, with membrane-mediated transmission traditionally attributed to EnVs. However, the composition and characteristics of viral populations encapsulated within extracellular vesicles (EVs) which are phospholipid bilayer nanoparticles released by all living organisms remain poorly understood. Here, we applied a phosphatidylserine (PS)-affinity enrichment strategy to isolate EV-encapsulated viral populations from human stool-derived extracellular viral-like particles (VLPs). Quantitative particle analysis revealed that EnVs exhibited an 11-fold higher PS affinity compared to free non-EnVs (fold change 2.79 vs 0.25). Metagenomic analysis revealed significant enrichment of non-EnVs within PS-positive fractions, including DNA viruses Salasmaviridae (3.84 ± 6.44 %) and RNA bacteriophage Fiersviridae (44.99 ± 32.80 %). Predicted Host-virus correlation analysis highlighted strong correlations between viral families Autographiviridae, Microviridae and host family Enterobacteriaceae. Functional annotation further showed enrichment of structural and replication-related genes in the EV-associated virome. These findings provide evidence for EVs-mediated encapsulation of non-EnVs, challenging the traditional dichotomy of viral classification. This noteworthy observation positions EVs encapsulation as a critical determinant in viral life cycles and underscores the need to revisit current viral taxonomy systems.