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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 丙型肝炎病毒(丙型肝炎病毒)是一个世界性的公共卫生问题，因为超过3%的人口感染了这种病毒，到目前为止治疗效果不佳。因此，有必要为开发更有效的药物确定新的目标。丙型肝炎病毒核心蛋白(CP)是病毒繁殖所必需的，参与多种病毒和细胞过程，被认为是一个重要的靶点。成熟的核心蛋白有179个氨基酸残基，而C端截短的HCVCP形式有124个残基，足以在体外组装。丙型肝炎病毒的组装机制尚不清楚。在这里，我们表达了C末端截短的HCVCP及其GFP(绿色荧光蛋白)融合形式，以研究在非特异性PolyGC DNA存在下的体外组装，使用电子显微镜、分光光度法、量热法和凝胶位移分析，在这种情况下是FCS。在没有和存在PolyGC DNA的情况下，用电子显微镜观察了这两种形式的核衣壳样颗粒(NLP)的形成。我们的数据还表明，通过分光光度法测量，NLP的形成依赖于蛋白质和PolyGC的浓度。等温滴定量热法数据表明，PolyGC DNA结合和刺激蛋白质-蛋白质相互作用的方式类似于这两种形式。为了研究组装过程中中间体的存在，我们使用了丙型肝炎病毒核心蛋白与GFP或AlexA标记的DNA分子的融合和FCS技术。FCS测量使我们能够通过形成的络合物的扩散或亮度的差异来识别中间体。这些测量将有助于我们识别可能的中间体，并了解病毒颗粒组装过程中的协同性。

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.