Molecular insights into the purified core protein of hepatitis C virus and the roles of its basic and hydrophobic amino acid clusters.

Hepatitis C virus (HCV) has been a global health concern for several decades and is known to cause severe liver diseases, such as cirrhosis and hepatocellular carcinoma. While the core protein, a major capsid component, plays essential roles in viral replication and pathogenesis, its detailed structural organization and domain-specific functions remain incompletely understood. Previously, we developed a method to purify the HCV core protein from bacterial cells under non-denaturing conditions and characterized its in vitro properties. In this study, we aimed to investigate the domain architecture and functional interactions of the HCV core protein. Small-angle X-ray scattering analysis of the purified core protein revealed that the HCV core consists of two domains. The smaller domain aligned with a model of two α-helices folded at an angle of 53.52° (0.93 rad), and the larger domain is associated with the basic N-terminal region. Further analysis of cellularly expressed and in vitro-purified core proteins containing mutations in the N-terminal basic or C-terminal hydrophobic amino acid clusters was performed using gel filtration and confocal microscopy. These analyses confirmed previous findings that the N-terminal region mediates viral genome binding, whereas the C-terminal region is involved in lipid membrane association. A pull-down assay also demonstrated a interaction between the N terminal HCV core protein and B23, a nucleolar protein known to be a core-binding partner. These findings provide new structural and functional insights into the HCV core protein, contributing to a deeper understanding of its role in viral replication and the molecular mechanisms underlying HCV pathogenesis.