The dengue virus (DENV) exploits host cell exosome pathways to disseminate and evade immunity. However, the host factors enabling this process remain poorly defined. Here, we demonstrate that DENV infection robustly induces expression of the short isoform of Reticulon 3 (RTN3S) in hepatic (Huh7) and monocytic cells, and that RTN3S is a critical driver of infectious exosome biogenesis. RTN3S physically associates with double-stranded viral RNA and the DENV non-structural protein 3 (NS3) in infected cells, indicating its integration into the viral replication complex. Loss of RTN3 markedly reduced exosome production and the exosomal export of viral RNA and proteins, demonstrating that RTN3S is required for efficient exosome-mediated viral release. Conversely, overexpression of full-length RTN3S dramatically increased the release of infectious virus-containing exosomes; truncation of the RTN3S C-terminal domain abolished this enhancement, confirming the essential role of the C-terminus in RTN3S’s pro-viral exosomal function. In DENV-infected monocytes, we observed a shift toward a CD16-positive intermediate phenotype, accompanied by the upregulation of genes involved in vesicle biogenesis and stress response. These infected monocytes also secreted higher levels of inflammatory cytokines. Similarly, monocytes from Dengue patients exhibited high RTN3 expression, which correlated with an expansion of intermediate (CD16⁺) subsets and enriched expression of vesicle trafficking machinery genes. These findings reveal a previously unrecognized mechanism by which DENV hijacks RTN3S to promote the formation of infectious exosomes, thereby facilitating viral dissemination and immune evasion. RTN3S thus represents a novel element of the Dengue pathogenesis and a potential target for host-directed antiviral strategies.

Dengue virus (DENV) continues to pose a major global health burden, yet therapeutic options remain limited due to the virus's capacity for immune evasion, serotype variability, and persistence. While exosomes have been implicated as vehicles for viral dissemination and immune evasion, the cellular mechanisms underlying their generation during DENV infection remain poorly defined. Here, we identify the endoplasmic reticulum (ER)-associated host protein Reticulon 3 (RTN3), particularly its short isoform RTN3S, as a critical regulator of replication-competent viral cargo loading during infectious exosome biogenesis in DENV infection. Using hepatic and monocytic cell models, we revealed that RTN3S expression is induced upon infection and that RTN3S directly associates with DENV replication complexes, facilitating the packaging of replication-competent viral RNA and host proteins into infectious exosomes. Loss of RTN3 function via CRISPR-Cas9 markedly attenuated exosome production and reduced the transfer of infectious viral components to recipient naïve cells. Mutational analyses of RTN3S further revealed that both its N-terminal amphipathic and C-terminal domains are essential for exosomal loading of viral material. Single-cell RNA-sequencing of peripheral blood mononuclear cells (PBMCs) from DENV-infected individuals confirmed RTN3 upregulation in monocytes, particularly in those displaying intermediate/classical phenotypes, and revealed a transcriptional signature linking RTN3 to ER stress, vesicle trafficking, and impaired antiviral responses. These findings uncover a previously unrecognized RTN3-centered mechanism by which DENV hijacks the host exosomal machinery to propagate infection and potentially escape immune surveillance. Thus, our findings demonstrate a novel function for RTN3 in orchestrating the biogenesis of infectious exosomes, providing mechanistic insight and identifying a new therapeutic axis for combating flavivirus infections through host-directed approaches.