Abstract
Zika virus (ZIKV) is a positive-sense single-stranded RNA virus that infects humans and can cause birth defects and neurological disorders. Its non-structural protein 3 (NS3) contains a protease domain and a helicase domain, both of which play essential roles during the viral life cycle. However, it has been shown that ZIKV NS3 has an inherently weak helicase activity, making it unable to unwind long RNA duplexes alone. How this activity is stimulated to process the viral genome and whether the two domains of NS3 are functionally coupled remain unclear. Here, we used optical tweezers to characterize the RNA-unwinding properties of ZIKV NS3-including its processivity, velocity, and step size-at the single-molecule level. We found that external forces that weaken the stability of the duplex RNA substrate significantly enhance the helicase activity of ZIKV NS3. On the other hand, we showed that the protease domain increases the binding affinity of NS3 to RNA but has only a minor effect on unwinding per se. Our findings suggest that the ZIKV NS3 helicase is activated on demand in the context of viral replication, a paradigm that may be generalizable to other flaviviruses.