对病毒复制基因改变的热力学和动力学研究B型肝炎病毒衣壳

StephenW

Genetically Altering the Thermodynamics and Kinetics of Hepatitis B Virus Capsid Assembly Has Profound Effects on Virus Replication in Cell Culture

    Zhenning Tan a,
    Megan L. Maguire b,
    Daniel D. Loebb and
    Adam Zlotnick a

- Author Affiliations

    aMolecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USA
    bMcArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA

ABSTRACT

Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effects of AEfs, with fast assembly kinetics and a strong protein-protein association energy. Also, the mutant was resistant to exogenous AEfs. In cell culture, the V124W mutant behaved like a potent AEf: expression of HBV carrying the V124W mutant was defective for genome replication. Critically, the V124W mutant interfered with replication of wild-type HBV in a dose-dependent manner, mimicking AEf activity. In addition, the V124W mutant was shown to adopt a more compact conformation than that of the wild type, confirming the allosteric regulation in capsid assembly. These studies show that the heteroaryldihydropyrimidine (HAP) binding pocket is a promiscuous target for inducing assembly. Suppression of viral replication by the V124W mutant suggests that mutations that fill the HAP site are not a path for HBV to escape from AEfs.

StephenW

衣壳（核心）组件是必不可少的B型肝炎病毒（HBV）的复制。我们假设调整为最佳的病毒复制，而不是最大的装配，装配动力学和稳定性。大会效应（阿英法西）提出的小分子破坏这种平衡，通过不恰当地提高核心组件。的AEF结合的核心结构的指导下，我们设计了一个AEF结合的核心蛋白，V124W突变体的结构模拟。 V124W突变体在生化研究，概括了影响阿英法西，快速组装动力学和强大的蛋白质 - 蛋白质协会能源。此外，该突变体抵抗外源性阿英法西。 V124W突变体在细胞培养中，表现得就像一个强有力的AEF：HBV携带V124W突变体的表达是有缺陷的基因组复制。更重要的是，V124W突变的干扰以剂量依赖的方式与野生型HBV的复制，模仿AEF活动。此外，V124W突变体显示出比野生型，确认在衣壳的装配的变构调节采用一个更紧凑的构象。这些研究表明，诱导组装的heteroaryldihydropyrimidine（HAP）结合口袋是一个混杂的目标。抑制病毒复制的V124W突变的突变，填补了HAP网站是不是乙肝病毒逃避阿英法西的路径。