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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 丙型肝炎病毒（HCV）是一个全球公共卫生问题，因为超过3％的人群感染了该病毒，疗法迄今为止效率低下。因此，有必要确定开发更有效药物的新目标。 HCV核心蛋白（CP）被描述为重要的靶标，因为它对于病毒传播至关重要，并且与几种病毒和细胞过程有关。成熟的核心蛋白呈现179个氨基酸残基，而C末端截短的HCVCP形式呈现124个残基，足以在体外组装。 HCV组装的机制尚不清楚。在这里，我们表达了C末端截短的HCVCP及其GFP（绿色荧光蛋白）融合形式，以便在使用电子显微镜，分光光度定律，量热测定和凝胶移位分析以及这种情况下，在非特异性多型多GDNA的情况下研究体外组装。在不存在polyGC DNA的情况下，通过电子显微镜观察到核素状样颗粒（NLP）的形成。我们的数据还表明，通过分光光度法测量，NLP的形成取决于蛋白质和PolyGC浓度。等温滴定量热法数据表明，多gc DNA结合并刺激蛋白质 - 蛋白质相互作用，类似于两种形式。为了研究组装过程中的间距的存在，我们使用了与GFP或Alexa标记为DNA分子和FCS技术的HCV核心蛋白。 FCS测量值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确定可能的中间体，并了解病毒颗粒组装过程中的合作性。