DDX3 regulates the cap-independent translation of the Japanese encephalitis virus via its interactions with PABP1 and the untranslated regions of the viral genome

The translation of global cellular proteins is almost completely repressed in cells with flavivirus infection, while viral translation remains efficient. The mechanisms of flaviviruses evade host translational shutoff are largely unknown. Here, we identified viral elements and host factors associated with JEV evasion of host shutoff. JEV 5′UTR lacked IRES or IRES-like activity, while noncapped 5′UTR initiated translation in the presence of 3′UTR. Furthermore, the elements DB2 and sHP-SL within 3′UTR were involved in the regulation of cap-independent translation, which is conserved in the genus Orthoflavivirus . By RNA affinity purification and mass spectrometry analysis, cellular DDX3 and PABP1 were identified as key factors in regulating cap-independent translation of JEV via their interactions with DB2 and sHP-SL RNA structures. Mechanistically, we revealed that DDX3 could bind to both 5′UTR and 3′UTR of the JEV genome to establish a closed-loop architecture, recruit eIF4G/eIF4A to form the DDX3/PABP1/eIF4G/eIF4A tetrameric complex via its interaction with PABP1, thereby recruiting 43S PIC to the 5′-end of the JEV genome to start translation. Our findings demonstrated a noncanonical translation strategy employed by JEV and further revealed the regulatory roles of DDX3 and PABP1 in this mechanism. These results expand our knowledge of the translation initiation regulation in flaviviruses under the state of host translational shutoff, which provides a conserved antiviral target against orthoflavivirus .