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

乙型肝炎病毒（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。病毒粒子由外包膜和内衣壳组成，外包膜由表面蛋白（HBs）密集包裹，内衣壳由乙型肝炎核心蛋白（HBc）形成。大多数包装核酸的包膜颗粒含有成熟的部分双链DNA基因组，该基因组是由衣壳内的前体RNA通过逆转录产生的。然而，没有基因组的磷酸化衣壳也很容易被包膜，这表明可能没有构象成熟信号使衣壳包膜胜任，但包膜的偏好是由包膜上游过程的动力学驱动的。在这里，我们想研究衣壳的结构性质，这是必不可少的包膜。到目前为止，我们已经证明，在病毒粒子中，衣壳通过突出的尖刺的尖端与包膜接触，而在重组衣壳中，干扰包膜的肽与这些尖端结合。这与其他使用突变筛选的人的发现形成对比，他们发现尖刺中心的疏水口袋对分泌表型很重要。这就引出了两个位点中哪一个对hbs结合更重要的问题。我们建议通过确定结合表面蛋白片段的重组衣壳的结构来解决这个问题。为此，我们将使用电子冷冻显微镜和图像处理，这使我们能够不受晶体接触的阻碍研究衣壳结构，分辨率高达2.4 Å。这些结构将显示尖刺的尖端、尖刺中心的口袋或两者是否与表面蛋白质片段结合。以这种方式询问具有特定包膜表型和磷酸化空衣壳的不同HBc突变体作为包膜能力衣壳的模拟物，将显示HBs结合是否受到衣壳结构变化的影响。我们还将确定干扰包膜的肽结合的位置，以及它们是否与HBs竞争结合位点，还是更有可能影响包膜上游的过程。所有的结构研究都将通过使用等温滴定量热法和/或表面等离子体共振测量来量化结合常数来补充，以测试结合常数的变化是否可能调节包络。基于我们最近的wt -衣壳结构和一个过早包膜突变体，我们预计在2.4-3 Å范围内的分辨率可以很容易地实现。因此，该项目将为临床相关的HBc突变体及其与Hbs的相互作用提供丰富的结构信息。结构信息将为包膜机制提供信息，并将作为识别能够干扰这一过程从而影响病毒成熟的硅分子的知识基础。

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.