Mechanical regulation of the helicase activity of Zika virus NS3 (Cao et al., Biophys J 2022)
Single-molecule characterization of the Zika virus NS3 helicase. (A) A representative recording and zoom-in views of RNA unwinding events driven by NS3h with 1 mM ATP at 18 pN of force. (B) Representative traces of RNA unwinding by NS3F at 0.1 mM ATP under 18-pN force. Raw data (blue points) are overlaid with the idealized stepping trace (red line).
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. Previous biochemical data showed that ZIKV NS3 has an inherently weak helicase activity, unable to unwind long RNA duplexes. How its activity is stimulated to process the viral genome and whether the two domains of NS3 are functionally coupled remain unclear. In collaboration with the Jin Lab, we use optical tweezers to characterize the RNA unwinding properties of ZIKV NS3—including its processivity, velocity, and step size—at the single-molecule level. We find that external forces that weaken the stability of the duplex RNA substrate significantly enhance the helicase activity of ZIKV NS3. On the other hand, the protease domain increases the binding affinity of NS3 to RNA but has only a minor effect on unwinding. Our findings suggest that ZIKV NS3 is activated on demand in the context of viral replication, a feature that mirrors that of SARS-CoV-2 nsp13 (Mickolajczyk et al. 2021) and may be generalizable to other flaviviruses.