A NEW PERSPECTIVE ON THE CHARACTERIZATION OF HEPATITIS C VIRUS CORE PROTEIN ASSEMBLY AND ITS INTERACTION WITH THE TUMOR SUPPRESSOR PROTEIN P53

About 50 million individuals worldwide are infected with the Hepatitis C Virus (HCV), and many progress to cirrhosis, fibrosis, hepatocellular carcinoma, and death. Although therapies have recently advanced, highlighting the need for a better understanding of the mechanisms involved in HCV replication and viral pathogenesis. To characterize HCV assembly and pathogenesis mechanisms, we investigated the structure of the HCV core protein (HCVcp), its cellular localization, and its interaction with the p53 tumor suppressor protein. Here, we used eukaryotic cell culture and Escherichia coli expression systems for the biophysical characterization of the interaction between 124 amino acids truncated form of HCVcp (C124) and the p53 DNA-binding domain (p53DBD), as well as the full-length p53 protein (p53full). Mobility assays, Nuclear Magnetic Resonance (NMR), spectrophotometric turbidity assays, Transmission Electron Microscopy (TEM), Bright-Field Microscopy and Förster Resonance Energy Transfer (FRET) were employed. Precipitation-based binding assays showed that, unlike p53DBD, C124 interacts more specifically with full-length p53, indicating the importance of the C-terminal region for this interaction. 1H/ 15N HSQC NMR spectra of C124 showed chemical shift perturbations in the presence of p53DBD or p53full, likely induced by interaction. C124 assembled into nucleocapsid-like particles in the presence of p53 consensus DNA and p53, as demonstrated by increased turbidity, TEM, and fluorescence spectroscopy. Our results also indicate that the C124–p53 interaction leads to aggregate formation, which is currently under investigation. HCVcp fused to green fluorescent protein (GFP) and p53 fused to DsRed fluorescent protein were co-expressed in Huh7 liver cells. Bright-field microscopy was able to evaluate the LLPS formation by HCVcp in presence of DNA, PEG and ACDAN in vitro and in hepatocytes. Acceptor photobleaching FRET analysis showed that C124 and p53 co-localize into the nucleus; however, no FRET phenomenon was observed so far under the initial conditions tested. Our data reveals a new perspective on nucleocapsid assembly and the HCVcp-p53 interaction, which may contribute to a better understanding of HCV infection.