Abstract

To identify novel host factors essential for orthoflavivirus replication, we performed proteomic profiling of endoplasmic reticulum fractions isolated from cells infected with Dengue virus or Zika virus (ZIKV). Among the enriched proteins, the splicing factor U2AF2 and its heterodimeric partner U2AF1 were found to be critical for efficient viral infection, functioning at the viral protein synthesis stage. The arginine/serine-rich (RS) domain and the first zinc knuckle (Zn1) of U2AF1, as well as nearly all domains except the RS domain of U2AF2, were essential for their proviral function. Disruption of the U2AF1–U2AF2 interaction potently suppressed ZIKV infection. U2AF1 and U2AF2 partially localize to the cytoplasm, and notably, the cytoplasmic U2AF2 was predominantly a truncated form that was sufficient to support viral replication. Both U2AF1 and U2AF2 bind to viral RNA, with U2AF1 binding being dependent on U2AF2. Interestingly, trans -complementation of the Aedes aegypti homolog u2af38 into U2AF1-knockout cells restored ZIKV replication, whereas expressing u2af50 in U2AF2-knockdown cells did not. However, knockdown of either u2af38 or u2af50 significantly inhibited the flavivirus replication in mosquitoes. Together, these findings reveal that flaviviruses co-opt host splicing factors in both human cells and mosquitoes, underscoring a conserved cross-species mechanism of viral exploitation.