Interaction of Hepatitis B capsid like particles with surface protein fragments and peptides interfering with envelopment

乙型肝炎衣壳样颗粒与表面蛋白片段和干扰包膜的肽的相互作用

• 批准号: 424878840
• 负责人: Professorin Dr. Bettina Böttcher
• 金额: --
• 依托单位: Lehrstuhl für Biochemie I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/424878840?language=en
• 关键词: Interaction; Hepatitis; capsid; like; particles

Hepatitis B Virus (HBV) is a major human pathogen with more than 250 million chronic carriers worldwide. The virions consist of an outer envelope with a membrane that is densely packed with surface proteins (HBs), and an inner capsid, formed by the hepatitis B core protein (HBc). The majority of enveloped particles with packaged nucleic acid contain a mature partly double stranded DNA genome that was generated from a precursor RNA by reverse transcription inside the capsids. However, phosphorylated capsids without a genome are also readily enveloped suggesting that there might be no conformational maturation signal that makes capsids envelopment competent but that preferences for envelopment are driven by the dynamics of the processes upstream of envelopment. Here we want to investigate the structural properties of the capsid that are essential for envelopment. So far, we have shown that in virions the capsid contacts the envelope via the tips of protruding spikes and that peptides that interfere with envelopment bind to these tips in recombinant capsids. This contrasts finding by others who have used mutational screens that identified a hydrophobic pocket in the center of the spikes as being important for the secretion phenotype. This leads to the question which of the two sites is important for HBs-binding. We propose to address this question by determining the structure of recombinant capsids with bound surface protein fragments. For this we will use electron cryo microscopy and image processing, which allows us to study the capsid structure unhindered by crystal contacts with resolutions of up to 2.4 Å. The structures will show whether the tips of the spikes, the pocket in the center of the spikes or both sites bind to the surface protein fragments. Interrogating different HBc mutants with specific envelopment phenotypes and phosphorylated empty capsids as mimics for envelopment competent capsids in such a way will show whether HBs binding is affected by changes in the capsid structure. We will also determine where peptides that interfere with envelopment bind and whether they compete with HBs for binding sites or are more likely to affect processes upstream of envelopment. All structural studies will be complemented by quantifying binding constants using isothermal titration calorimetry and/or surface plasmon resonance measurements to test whether changes in the binding constant are likely to modulate envelopment.Based on our recent structures of WT-capsids and a premature envelopment mutant, we expect that resolutions in the range of 2.4-3 Å can be readily achieved. Therefore, this project will provide a wealth of structural information on clinically relevant HBc mutants and their interaction with Hbs. The structural information will inform on the envelopment mechanism and will serve others as knowledge base for identifying molecules in silico that can interfere with this process and thus with viral maturation.

B型肝炎病毒（HBV）是一种主要的人类病原体，全世界有超过2.5亿慢性携带者。病毒体由具有密集填充有表面蛋白（HB）的膜的外被膜和由B型肝炎核心蛋白（HBc）形成的内衣壳组成。大多数具有包装核酸的包膜颗粒含有成熟的部分双链DNA基因组，其通过衣壳内的逆转录由前体RNA产生。然而，没有基因组的磷酸化衣壳也很容易被包裹，这表明可能没有构象成熟信号使衣壳能够进行修饰，但是修饰的偏好是由修饰上游过程的动力学驱动的。在这里，我们想研究的衣壳的结构特性是必不可少的。到目前为止，我们已经表明，在病毒粒子的衣壳接触包膜通过突出的刺突的尖端和肽干扰结合到重组衣壳的这些尖端。这与使用突变筛选的其他人的发现形成对比，这些突变筛选将刺突中心的疏水口袋鉴定为对分泌表型很重要。这就引出了一个问题，即这两个位点中的哪一个对β-结合很重要。 我们建议解决这个问题，通过确定与结合的表面蛋白片段的重组衣壳的结构。为此，我们将使用电子冷冻显微镜和图像处理，这使我们能够研究不受晶体接触阻碍的衣壳结构，分辨率高达2.4 μ m。这些结构将显示刺突的尖端、刺突中心的口袋或这两个位点是否与表面蛋白片段结合。以这种方式询问具有特异性表达表型的不同HBc突变体和作为表达能力衣壳的模拟物的磷酸化空衣壳将显示HBs结合是否受衣壳结构变化的影响。我们还将确定干扰补体结合的肽的位置，以及它们是否与HB竞争结合位点或更可能影响补体上游的过程。所有的结构研究将通过定量结合常数使用等温滴定量热法和/或表面等离子体共振测量来补充，以测试结合常数的变化是否有可能调制pacement.Based上我们最近的WT-衣壳和过早pacemment突变体的结构，我们预计，分辨率在2.4-3 μ m的范围内可以很容易地实现。因此，该项目将提供丰富的临床相关HBc突变体及其与Hbs相互作用的结构信息。结构信息将提供有关病毒成熟机制的信息，并将作为其他人的知识基础，用于在计算机上识别可能干扰该过程并因此干扰病毒成熟的分子。