HEPATITIS C VIRUS CORE PROTEIN-DNA INTERACTIONS: IN VITRO ASSEMBLY ANALYSES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hepatitis C virus (HCV) is a worldwide public health problem because more than 3% of the population is infected by this virus 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 (CP) has been described as an important target because its is essential for viral propagation and its 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 to assemble in vitro. The mechanisms of HCV assembly are not well understood. Here we express the C-terminal truncated HCVCP and its GFP (green fluorescent protein) fused form in order to investigate the in vitro assembly in the presence of a nonspecific polyGC DNA using electron microscopy, spectrophotometry, calorimetry and gel shift assay and in this case FCS. The formation of nucleocapsid-like particles (NLPs) was observed by electron microscopy for both forms in the absence and in the presence of polyGC DNA. Our data also showed that the formation of NLPs was dependent of protein and polyGC concentration, as measured by spectrophotometry. Isothermal titration calorimetry data have shown that polyGC DNA binds and stimulate protein-protein interactions similarly to both forms. In order to investigate the presence of intermidiates of the assembly process we used the HCV core protein fused to GFP or Alexa labeled DNA molecules and FCS techniques. FCS measurements allow us to identify intermediates either by the difference in diffusion or brightness of the complexes formed. These measurements will help us to identify the possible intermediates and to understand cooperativity in the viral particle assembly process.