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

丙型肝炎病毒核心蛋白-DNA 相互作用：体外组装分析

• 批准号: 7956533
• 负责人: Andre Oliveira
• 金额: $ 1.05万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956533
• 关键词: Amino Acids; C-terminal; Calorimetry; Cell physiology; Complex; Computer Retrieval of Information on Scientific Projects Database; Core Protein; DNA; DNA Binding; Data; Development; Diffusion; Electron Microscopy; Electrophoretic Mobility Shift Assay; Fluorescence; Funding; Grant; Green Fluorescent Proteins; Hepatitis C virus; In Vitro; Institution; Label; Laboratories; Measurement; Measures; Nucleocapsid; Pharmaceutical Preparations; Population; Process; Proteins; Public Health; Research; Research Personnel; Resources; Source; Spectrophotometry; Techniques; Titrations; United States National Institutes of Health; Viral; Virus; Virus Assembly; hepatitis C virus nucleocapsid protein; particle; protein protein interaction

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 丙型肝炎病毒 (HCV) 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，且迄今为止治疗方法效率低下。因此，需要确定新的靶点来开发更有效的药物。 HCV 核心蛋白 (CP) 被描述为一个重要的靶标，因为它对于病毒传播至关重要，并且参与多种病毒和细胞过程。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。 HCV 组装的机制尚不清楚。在这里，我们表达 C 端截短的 HCVCP 及其 GFP（绿色荧光蛋白）融合形式，以便使用电子显微镜、分光光度法、量热法和凝胶位移测定法研究非特异性 PolyGC DNA 存在下的体外组装，在本例中燃料电池系统。通过电子显微镜观察在不存在和存在 PolyGC DNA 的情况下两种形式的核衣壳样颗粒 (NLP) 的形成。我们的数据还表明，NLP 的形成依赖于蛋白质和聚 GC 浓度（通过分光光度法测量）。等温滴定量热法数据表明，polyGC DNA 与两种形式类似地结合并刺激蛋白质-蛋白质相互作用。为了研究组装过程中中间体的存在，我们使用了与 GFP 或 Alexa 标记的 DNA 分子融合的 HCV 核心蛋白和 FCS 技术。 FCS 测量使我们能够通过形成的复合物的扩散或亮度差异来识别中间体。这些测量将帮助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。