Single-molecule-binding studies of antivirals targeting the hepatitis C virus core protein

Abstract

ABSTRACT The hepatitis C virus (HCV) core protein (HCVcp) is the most highly conserved protein encoded by the HCV genome, and its N-terminal domain ( NTD HCVcp) plays a crucial role in nucleocapsid assembly. Together, these two features make it an attractive target for antiviral therapeutics. However, NTD HCVcp is intrinsically disordered, leading to a high degree of conformational heterogeneity, and given its essential role in nucleocapsid assembly, it also tends to oligomerize at high concentrations, both of which make it difficult to characterize heterotypic intermolecular interactions between monomeric NTD HCVcp and potential therapeutics using conventional structure-based approaches. Here, we use single-molecule FRET spectroscopy to overcome these challenges and study the structural and energetic aspects of binding interactions involving different viral genotypes of NTD HCVcp and antiviral therapeutics based on antibodies, aptamers, peptides, and small molecules. Our findings highlight distinct binding mechanisms associated with these molecular interactions. For example, binding of high-affinity antibodies does not perturb the end-to-end distance of NTD HCVcp and is weakly impeded by electrostatic repulsions between similarly charged residues. Conversely, the low-nanomolar equilibrium dissociation constants of antiviral DNA aptamers arise from strong attractive electrostatic interactions that greatly decrease the end-to-end distance of NTD HCVcp. Furthermore, low-affinity antiviral peptides promote oligomerization of NTD HCVcp. Finally, the small-molecule antiviral compound we studied does not appear to affect any of our experimental observables, suggesting that binding may not alter the conformational properties of NTD HCVcp. IMPORTANCE The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.