MON-313 Viral Envelope Glycoproteins Incorporate into Extracellular Vesicles by Hijacking Endogenous Pathways of Exosome Biogenesis in Human Cells

Abstract Disclosure: D. Levy: None. M. Do: None. J. Zhang: None. C. Olson: None. Z. Ehlinger: None. R. Flojo: None. J. Ku: None. K. Asano: None. D. Diebold: None. H. Afzali: None. A. Wang: None. B. Lu: None. Exosomes, nanoscale extracellular vesicles (EVs), are emerging as versatile platforms for targeted drug delivery and precision medicine. Recent studies suggest that viral envelope glycoproteins (GPs) can integrate into exosomal membranes, enhancing tissue specificity and cellular uptake. However, the molecular mechanisms underlying this process remain poorly understood. To address this gap, we fused viral GPs with green fluorescent protein (GFP) and expressed them in cultured human cells. Using confocal microscopy, we tracked the incorporation of GPs into exosomes. Our findings demonstrate that all five tested viral envelope GPs, derived from both DNA and RNA viruses, successfully integrated into exosomal membranes in transient and stable cell models. Molecular imaging revealed that viral GPs trafficked through endocytic compartments and co-localized with exosomal markers CD63 and XPACK, indicating that they hijacked endogenous exosome biogenesis pathways for integration. Further analysis showed that deletion of the GP ectodomain did not disrupt their incorporation into exosomes, confirming that while the ectodomain governs cell tropism, it is dispensable for exosome integration. Functional assays demonstrated that exosomes modified with vesicular stomatitis virus glycoprotein (VSVG) exhibited significantly enhanced uptake by recipient cells compared to unmodified exosomes, validating their functionalization. Collectively, these findings elucidate the mechanisms by which viral envelope GPs exploit exosome biogenesis pathways for membrane integration. This discovery advances our understanding of the shared biogenesis mechanisms between exosomes and viral-like particles (VLPs) and provides a novel approach for engineering exosome-based drug delivery systems with enhanced targeting and uptake capabilities. Presentation: Monday, July 14, 2025