Molecular Modulation of HBV Capsid Assembly

描述(申请人提供)：乙肝病毒(乙肝)是一种带有单链RNA中间体的包膜双链DNA病毒。复制需要正确组装乙肝病毒的二十面体核心；RNA前基因组的逆转录在完成的衣壳内进行，即核心的蛋白质外壳。目前的抗乙肝治疗药物是干扰素和核苷类似物的乙肝病毒逆转录酶(POL)。这两种方法的效果都有限，而且成本可能很高。像任何单一疗法一样，POL抑制剂选择耐药突变；其中一些突变逃脱了疫苗，因为POL和表面蛋白基因重叠。我们主张将衣壳组装作为抗病毒治疗的补充靶点。组装对乙肝病毒复制至关重要，并且是病毒独有的(没有衣壳蛋白的细胞同源)。乙肝病毒衣壳由120个蛋白质二聚体组成。在体外，一些小分子和盐可以诱导组装。我们发现，乙肝病毒的装配是受变构调控的：衣壳蛋白有装配活性构象和非装配活性构象。因此，有利于非活性形式的小分子将抑制组装；有利于活性形式的分子将加速这一过程。我们已经发现，一些分子误导衣壳组装，推测是通过扭曲二聚体的几何结构，导致畸形和无功能结构的快速产生。目前，我们在每个类别中都有一小部分分子可供选择。在这项提案中，我们描述了一种开发组装导向抗病毒药物的策略。从预先存在的先导开始，我们将研究最活跃分子改变组装的物理和结构基础。利用这些信息，我们将利用最近在组装量化方面的进展来筛选候选小分子文库，以增强组装加速、抑制和误导的活性。这些分子的作用也将在表达乙肝病毒的培养细胞中进行测试。 超过3.5亿人患有慢性乙肝病毒(乙肝)感染，其中包括超过125万美国人。在全球范围内，今年乙肝病毒将导致100万人死亡。目前的抗病毒策略主要集中在病毒酶上。我们提出了一种新的策略来开发针对病毒组装的抗病毒分子。

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) is an enveloped dsDNA virus with a ssRNA intermediate form. Correct assembly of the icosahedral core of HBV is required for replication; reverse transcription of the RNA pre-genome takes place within the completed capsid, the protein shell of the core. Current anti-HBV therapeutics are interferon and nucleoside analog inhibitors of HBV reverse transcriptase (Pol). Both approaches have limited efficacy and can be expensive. Like any monotherapy, Pol inhibitors select for resistant mutants; some of these mutations escape the vaccine because Pol and surface protein genes overlap. We advocate capsid assembly as a complementary target for antiviral therapy. Assembly is critical to HBV replication and is unique to the virus (there are no cellular homologs to capsid protein). The HBV capsid is constructed from 120 protein dimers. In vitro, assembly can be induced by a number of small molecules and salts. We have found that HBV assembly is allosterically regulated: capsid protein has assembly-active and -inactive conformations. Consequently, small molecules that favor the inactive form will inhibit assembly; molecules that favor the active form will accelerate the process. We have found that some molecules misdirect capsid assembly, presumably by distorting the dimer geometry, giving rise to the rapid production of misshapen and nonfunctional structures. At this time, we have a small selection of molecules in each category. In this proposal we describe a strategy for developing assembly-directed antivirals. Starting with the pre-existing leads, we will investigate the physical and structural basis for altered assembly by the most active molecules. Using this information, we will then take advantage of recent advances in the quantification of assembly to screen candidate small-molecule libraries for enhanced activity in assembly acceleration, inhibition, and misdirection. The effects of these molecules will also be tested in cultured cells that express HBV. More than 350 million individuals suffer from chronic infection with hepatitis B virus (HBV), including more than 1.25 million Americans. Worldwide, HBV will contribute to 1 million deaths this year. Current antiviral strategies focus on virus enzymes. We propose a new strategy for developing antiviral molecules that target virus assembly.