Resumo

Hepatitis C is a worldwide public health problem since more than 3% of the population is infected by Hepatitis C virus (HCV), and therapies are inefficient so far. Thus, there is a need to identify new targets for the development of more efficient drugs. The HCV core protein (HCVCP) has been described as an important target because it is essential for viral propagation and is involved in several viral and cellular processes. Mature core protein presents 179 amino acid residues whereas the C-terminal truncated HCVCP form presents 124 residues that are enough for in vitro assembly of particles. The mechanisms of HCV assembly are not well understood. Here we express the C-terminal truncated HCVCP fused with GFP (green fluorescent protein) in order to investigate the in vitro assembly in the presence of nonspecific nuclei acids using electron microscopy, spectrophotometry, calorimetry, gel shift assay and fluorescence correlation spectroscopy (FCS). The formation of nucleocapsid-like particles (NLPs) was observed by electron microscopy in the absence and in the presence of nucleic acids, what demonstrated that the capacity of assembly is conserved after the GFP fusion. Our data also show that the formation of NLPs was dependent of protein and DNA concentrations, as measured by spectrophotometry. Isothermal titration calorimetry data have shown that the assembly is enthalpically driven. Gel shift assays show that no assembly intermediate form was observed for HCVCP, indicating a highly cooperative process. In addition to microscopy observations, FCS data also indicate the presence of NLPs in the absence of nucleic acids, what suggest that the free protein is in equilibrium with oligomeric forms. In conclusion, our results show that the formation of NLPs in vitro is not altered by the presence of GFP, since the assembly process remains cooperative, dependent on the protein and nuclei acid concentration and enthalpically favored. Thus, we suggest that interesting information can be obtained by expression of HCV core protein fused with GFP in human cells, since advanced methods in fluorescence spectroscopy could be applied. Those studies in human cells are in progress.