DUAL FUNCTION OF ZIKA VIRUS NS2B-NS3 PROTEASE

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of an NS2B polypeptide that associates with a proteolytic N terminal fragment of NS3 polypeptide (NS3pro) to form NS2B-NS3pro. The larger C-terminal fragment of NS3 polypeptide contains helicase activity. In the present study, we discovered that ZIKV NS2BNS3pro efficiently binds single-stranded (ss) RNA (Kd ~0.3 uM), suggesting that the protease may have a novel function. We tested an array of NS2B-NS3pro modifications and found that NS2B NS3pro constructs that adopt the recently discovered super-open conformation could not bind ssRNA. Likewise, stabilization of NS2B-NS3pro in the closed (proteolytically active) conformation by substrate-like inhibitors abolished ssRNA binding. Therefore, we suggest that ssRNA binding occurs when ZIKV protease adopts the open conformation, which could be modeled using dengue NS2B-NS3pro in the open conformation. ssRNA binding competes with ZIKV NS2B-NS3pro protease activity, likely by shifting the complex into the open conformation. Modeling of ZIKV NS3 helicase activity based on homologous crystal structures suggests that the open conformation of NS3pro domains provides a positively charged surface contiguous with the NS3 helicase domain. Such a positively charged surface is well poised to bind ssRNA, providing an explanation for the previously observed requirement of NS3pro for RNA processivity by viral helicase. Our structure-function analyses suggest that binding of ssRNA by the protease domain of NS3 is likely to be a universal feature of Flaviviridae, given the high level of homology between NS3 protease-helicase proteins in this family.