Characterization of lipid binding motifs of Hepatitis C Virus Non-structural Protein 5A

Hepatitis C virus (HCV) remains a significant global health challenge, affecting approximately 50 million people worldwide, with about one million new infections occurring annually. The virus has a considerable disease burden, contributing to an estimated 242,000 deaths in 2022. HCV belongs to the Flaviviridae family and contains a single-stranded RNA genome. The virus undergoes a multi-step lifecycle that includes viral entry into host cells, translation of its RNA into viral polyprotein, replication of its genome, and the assembly of new virus particles. A critical component of the viral replication and assembly machinery is the non- structural protein 5A (NS5A). NS5A is composed of an N-terminal amphipathic helix (AH) and three distinct domains. Domain 1 (D1) is crucial for RNA binding and dimerization, while domains 2 (D2) and 3 (D3) remain less well- characterized, although they are known to contribute to the overall function of the protein. One of the key features of NS5A is its ability to interact with host lipids, especially phosphatidylinositol (PI) derivatives and phosphatidic acid (PA). These interactions are essential for the formation of the membranous web (MW), a network of altered host cell membranes serving as a platform for viral replication. This study aimed to characterize specific lipid-binding regions within NS5A. To this end, site-directed mutagenesis was employed to introduce targeted mutations in three potential lipid-binding clusters (clusters I-III) within full-length (FL) or truncated NS5A constructs. The primary objective was to investigate how these mutations impact NS5A's ability to bind negatively charged phospholipids, particularly phosphoinositides (PIPs) and PA, and how this might affect the protein's architecture. To conduct these experiments, NS5A variants were expressed in insect cells using a baculovirus-mediated infection system. This system allowed the production of several NS5A constructs, including FL NS5A, as well as deletion mutants lacking D2 and D3 (Δ2-3), the amphipathic helix (ΔAH), or both (ΔAH-Δ2-3). Once purified, these proteins were subjected to Protein-Lipid Overlay Assays (PLOA) to evaluate their lipid-binding properties. Thermal shift assays (TSA) were also performed to assess the impact of the introduced mutations on the stability of NS5A. By analyzing the protein's thermal denaturation profile, insights were gained into how these mutations might influence the overall architecture of the protein and its lipid-binding capabilities. This combination of biochemical assays allowed for a comprehensive analysis of NS5A's lipid-binding properties and the impact of specific mutations on its structure and function. The results of the PLOA confirmed a distinct lipid-binding pattern for wild-type (wt) NS5A and displayed strong binding to negatively charged phospholipids, particularly PI monophosphates and PA. Mutations in Cluster I, K68E, in both constructs, resulted in a significant decrease in lipid-binding affinity, although the specificity for certain lipids was retained. Cluster II mutants exhibited a binding profile similar to wt protein, with higher background staining. Cluster III mutants showed reduced binding compared to wild-type NS5A, but their binding was stronger than that observed for Cluster I mutants. These findings suggest that Cluster I plays a critical role in NS5A's lipid-binding; Cluster II is dispensable for the interactions, while Cluster III may have an intermediate phenotype. Further analysis using TSA provided additional insights into the structural consequences of these mutations. The TSA results indicated that the K68E mutation in Cluster I does not affect protein folding since the mutant protein retained its thermal stability, as evidenced by sigmoidal melting curves similar to those observed for wild-type NS5A. Interestingly, the absence of a second peak in the TSA after relipidation of the K68E mutant suggested that this mutation interferes with NS5A's ability to oligomerize, a process that may be crucial for its role in membrane remodeling. Overall, the findings of this study suggest that specific clusters of positively charged amino acids within NS5A, particularly Cluster I, are critical for the protein's lipid-binding properties. These results provide first insights into the molecular mechanisms by which NS5A interacts with host lipids and contributes to the formation of the HCV-induced MW.