Flavivirus NS1 possesses NTPase and RNA helix unwinding activities

Flaviviruses are a group of arthropod-borne positive-sense RNA viruses, including Zika virus (ZIKV), dengue virus 2 (DENV2) and Japanese encephalitis virus (JEV). For RNA viruses, RNA helicases play critical roles in viral replication by unwinding double-stranded RNA intermediates generated in viral life cycles by remodelling viral RNA structures. Here, we report that NS1 proteins from ZIKV, DENV2 and JEV, possesse NTPase and RNA helix unwinding activities. ZIKV NS1 unwinds dsRNA in an NTP-dependent manner with 5'→3' directionality, and its unwinding activity is broad-spectrum, targeting dsRNA, dsDNA and RNA/DNA hybrids. Moreover, both DENV2 and JEV NS1 also display NTPase and helicase activities, but they differ in substrate preference and directionality: JEV NS1 unwinds in the 5'→3' direction like ZIKV NS1, whereas DENV2 NS1 unwinds only shorter duplexes and shows 3'→5' directionality. Mutagenesis identified the basic residue at position 211 (arginine in ZIKV, lysine in DENV2/JEV) as critical for the unwinding activity of ZIKV and JEV NS1, but dispensable for DENV2 NS1. These findings indicate that flavivirus NS1 represents a previously unrecognized non-canonical RNA helicase, suggesting that NS1 may have the capacity to participate in RNA remodeling processes.