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Flavivirus infection requires cellular machinery to facilitate replication and spread. All known flaviviruses replicate in association with the host endoplasmic reticulum (ER), where genome replication is confined within virus-induced ER invaginations called viral replication organelles (vROs). Despite the central role of these structures during flavivirus infection, the mechanisms underlying vRO biogenesis remain undefined\u2014particularly the membrane rearrangements required for their formation. In this work, we report a conserved role for a cellular ER remodeling protein, atlastin-2 (ATL2), in the organization of vROs within infected cells. Using confocal and electron microscopy, we show that ATL2 depletion leads to a reduction in vRO spatial distribution in flavivirus-infected cells. Changes in vRO distribution corresponded with a decrease in virus production and robust induction of innate immune responses. We also demonstrate that ATL2 accumulates in areas of vRO formation during flavivirus infection. Critically, mutational analysis showed that a tethering-competent but fusion-defective ATL2 mutant was sufficient to rescue DENV and ZIKV replication in ATL2-knockout cells. Finally, targeting of ATL2 activity using synthetic peptides significantly reduced DENV replication in both immortalized and human primary cells, suggesting a possible avenue for targeting host ER functions to limit flavivirus replication. Taken together, these results show that membrane tethering plays a critical and conserved role in flavivirus infection, functioning to organize membranes for vRO biogenesis and limit cellular immune activation. Importantly, we provide evidence that ATL2-mediated membrane organization can be targeted to inhibit viral replication.\n                  <\/jats:p>","DOI":"10.1371\/journal.pbio.3003556","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T17:26:38Z","timestamp":1789493198000},"page":"e3003556","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Atlastin-2-mediated endoplasmic reticulum membrane tethering is critical for flavivirus replication"],"prefix":"10.1371","volume":"24","author":[{"given":"Jonathan Einterz","family":"Owen","sequence":"first","affiliation":[{"name":"School of Medicine, Emory University","place":["Atlanta, Georgia, United States of America"],"department":["Department of Microbiology and Immunology"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheyanne 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