| Summary: | Direct-acting antivirals (DAAs) targeting the nonstructural 5A (NS5A) protein of Hepatitis C virus (HCV) are essential drugs with their exceptional clinical success in actual patients. However, surprisingly, their modes of action (MoA) are still unknown, and even more importantly, resistant types of the virus to DAAs are also quickly emerging. Therefore, it is vital to better understand the behavior of NS5A in every aspect, which may facilitate the development of new classes of inhibitors or methods that improve the efficacy of currently available DAAs. To this end, we have employed a facile method based on an expression vector with an N-terminal SUMO tag and have successfully purified NS5A-domain I (DI), where most resistance mutations are mapped on so far. Our system enabled us to achieve soluble proteins in high yields, which allowed to investigate the structural dynamics of NS5A and self-association behavior in vitro. By employing dynamic light scattering (DLS), size-exclusion chromatography (SEC), and chemical crosslinking experiments, we have shown that NS5A-DI from genotype 1b of HCV intrinsically self-associates in solution at physiological conditions. Notably, they remain in an equilibrium state, where monomers, dimers (transient), and higher-order oligomers coexist as a heterogeneous mixture. Small angle X-ray scattering (SAXS) studies suggest that the dimer units of NS5A-DI assemble as a helical polymer to form higher order structures. Interestingly, NS5A-DI of genotype 1a exhibits an alternative form of dynamic equilibrium of the proteins in solutions. To understand its MoA, we have tested the effect of daclatasvir (DCV), the most prominent DAA, on self-association and membrane-anchoring behavior of the protein. We have analyzed the effect of DCV on NS5A using DLS and SAXS. Our results showed that DCV does not shift the dynamic self-association behavior of the protein. However, DCV triggered the aggregation of protein, which is consistent with the features observed earlier. The multimers of NS5A in the cell culture were detected by fluorescent tags covalently attached to NS5A, and the formation of larger foci of NS5A in the presence of NS5A inhibitors was reported. As the mechanism of HCV infection is membrane-associated, we also explored the possible effect of DCV on the membrane anchoring behavior of NS5A protein by utilizing quartz crystal microbalance with dissipation monitoring (QCM-D) on supported bilayer platforms. These studies demonstrated that DCV does not completely block the membrane-anchoring of domain I of NS5A. We have tested wild-type and DCV-resistant mutant NS5A-DI proteins and observed a slight difference in their membrane-anchoring behavior when incubated with DCV together. This study highlights the conformational variability of the NS5A domain 1 of HCV, which might be playing a significant role in the modulating of the multiple functions of the protein.
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