Molecular Modulation of HBV Capsid Assembly

描述（由申请人提供）：乙型肝炎病毒（HBV）是一种包膜dsDNA病毒，具有ssRNA中间形式。HBV二十面体核心的正确组装是复制所必需的；RNA前基因组的逆转录发生在完整的衣壳内，即核心的蛋白质外壳。目前的抗HBV治疗是干扰素和核苷类似物抑制剂的HBV逆转录酶（Pol）。这两种方法的效果都有限，而且可能很昂贵。像任何单一疗法一样，Pol抑制剂选择耐药突变体；由于Pol和表面蛋白基因重叠，其中一些突变逃过了疫苗。我们提倡衣壳组装作为抗病毒治疗的补充靶点。组装对HBV复制至关重要，并且是病毒所特有的（衣壳蛋白没有细胞同源物）。HBV衣壳由120个蛋白二聚体构成。在体外，组装可以由许多小分子和盐诱导。我们发现HBV的组装是变构调节的：衣壳蛋白具有组装活性和非组装活性构象。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会加速这一过程。我们发现一些分子可能通过扭曲二聚体的几何形状来误导衣壳的组装，从而导致畸形和非功能结构的快速产生。此时，我们在每个类别中都有一小部分分子。在这个建议中，我们描述了一种开发面向组装的抗病毒药物的策略。从预先存在的导联开始，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，我们将利用组装量化方面的最新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HBV的培养细胞中进行测试。

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.