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Among them, SAMD9 and its paralog SAMD9L have recently emerged as important antiviral effectors with translation-inhibitory activity. While both proteins restrict poxvirus, rotavirus and reovirus replication, only SAMD9L has been shown to inhibit HIV and other lentiviruses. In this study, we identify human SAMD9L as a potent and broad-spectrum restriction factor that targets multiple medically relevant flaviviruses, including West Nile virus (WNV), Zika virus (ZIKV), dengue virus (DENV), and Usutu virus (USUV). Exogenous expression of SAMD9L, but not SAMD9, efficiently suppressed replication of all tested flaviviruses. Furthermore, its knockdown in human myeloid cells, including microglial cells and primary macrophages, impaired the antiviral activity of type I interferon, identifying SAMD9L as a key antiviral ISG in primary target cells of flavivirus infection. Mechanistically, we demonstrate that SAMD9L inhibits viral replication by targeting the translation of flaviviral RNA, and that this activity depends on its Schlafen-like ribonuclease domain, previously implicated in the inhibition of HIV-1 translation. Interestingly, although SAMD9 does not inhibit flavivirus replication, it is able to repress the translation of flaviviral RNA outside the context of infection, suggesting that its activation may be virus-specific or that flaviviruses have evolved mechanisms to evade or counteract SAMD9\u2019s antiviral activity. Finally, we confirm that SAMD9 and SAMD9L overexpression induces activation of the innate immune response. However, this immunostimulatory function is dispensable for SAMD9L-mediated antiviral activity, since SAMD9L is able to restrict flavivirus replication independently of innate immune activation. Together, our findings broaden the known antiviral repertoire of SAMD9L, establish its essential role in restricting flavivirus replication via translational repression, and highlight its function as a key component of the cellular defenses against flaviviruses in myeloid cells.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013773","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T18:32:42Z","timestamp":1765218762000},"page":"e1013773","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":5,"title":["SAMD9L inhibits flavivirus translation independently of its capacity to trigger innate immune 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