Multimode Observation of Virus Capsid Assembly

 描述(申请人提供)：在大约一半的已知病毒家族中发现了自组装的二十面体衣壳。然而，组装机制仍然是一个黑匣子，尽管组装是生物学的基础。例如，组装对于细胞定位、核酸包装和新型组装导向的抗病毒药物(有时被描述为组装抑制剂，尽管它们实际上促进错误组装)是关键的。到目前为止，几乎所有的组装观察都依赖于结构不明确的复合体的集合。同样，几乎所有的组装模型都依赖于关于溶液中亚单位的状态、不完整衣壳中亚单位的状态以及参与组装的中间体列表的特别假设。我们提出了一个详细的分析乙肝病毒(乙肝病毒)的组装。涉及突变体和突变体组合的体外实验将通过与细胞培养中组装的比较来验证。基于建模研究，我们假设120-二聚体T=4乙肝病毒衣壳的组装将涉及相对较少的中间体。如果是这样的话，它将直接影响到 病毒RNA的组织，它在体内是在组装过程中包装的。在这项提案中，我们将使用生化技术和电子显微镜(Zlotnick，Wang Labs)、时间分辨SAXS(Raviv Lab)和电荷检测质谱仪(Jarrold Lab)的单颗粒观察来表征乙肝病毒衣壳的自组装。初步数据显示了实验的可行性，并表明了一些受欢迎的中间体的存在。这些结果将有助于开发忠实地复制组装的数学模型，并有助于识别干扰正常组装的显著点。乙肝病毒是人类最小的病原体之一(3200bpDNA基因组)，也是分布最广的病原体之一。每年约有3.5亿人患有慢性乙肝，导致约60万人死亡。目前的治疗方法抑制但不能治愈感染。几家制药公司正在研究乙肝病毒衣壳蛋白作为一种抗病毒靶标，有可能实现功能性治疗

 DESCRIPTION (provided by applicant): Self-assembling icosahedral capsids are found in about half of known virus families. Yet, the mechanisms of assembly remain a black box, even though assembly is fundamental to biology. For example, assembly is critical to, cellular localization, nucleic acid packaging, and new classes of assembly-directed antivirals (sometimes described as assembly inhibitors though they actually promote mis-assembly). To date, almost all observations of assembly have depended on ensembles of structurally ill-defined complexes. Similarly, almost all models of assembly have depended on ad hoc assumptions regarding the state of subunits in solution, the state of subunits in incomplete capsids, and the list of intermediates participating in assembly. We propose a detailed analysis of the assembly of Hepatitis B Virus (HBV). In vitro experiments, involving mutants and combinations of mutants will be validated by comparison to assembly in cell culture. Based on modeling studies we hypothesize that assembly of the 120-dimer T=4 HBV capsid will involve a relatively small number of intermediates. If this is the case it has immediate implications for the organization of viral RNA, which in vivo is packaged during the assembly process. In this proposal we will characterize HBV capsid self-assembly using biochemical techniques and electron microscopy (Zlotnick, Wang labs), time resolved SAXS (Raviv Lab), and single particle observations by charge detection mass spectrometry (Jarrold Lab). Preliminary data show the feasibility of experiments and indicate the presence of some favored intermediates. These results will contribute to the development of mathematical models that faithfully replicate assembly and can help identify salient points for interfering with normal assembly. HBV is one the smallest human pathogens (3200 bp DNA genome) and one of the most widespread. About 350 million people have chronic HBV leading to about 600,000 deaths each year. Current therapeutics suppress but do not cure infection. Several pharmaceutical companies are investigating HBV capsid protein as an antiviral target with potential to achieve a functional cure