Structural characterization of nucleoprotein cores of human adenoviruses

人腺病毒（HAdV）是一种大的（MW ~150 mDa）、复杂的、无包膜的双链DNA病毒，其引起急性呼吸道疾病、流行性角膜结膜炎和胃肠道感染。值得注意的是，腺病毒（AdV）被用作基因、疫苗和溶瘤病毒疗法的病毒载体。虽然在组装的（二十面体）病毒衣壳中的主要和次要衣壳蛋白（CP）的原位结构和排列已经在近原子分辨率下确定，但目前还没有关于核蛋白核心（包括线性双链DNA（~36 kbp）和“组蛋白样”核心蛋白）如何组织的信息。核心密度在AdV的二十面体重建中显示为涂抹球。这与在噬菌体和疱疹病毒中看到的dsDNA的特征性卷曲组织形成对比。核核心组织的细节为基因组包装成AdV的机制提供了线索，这一机制仍然没有得到解决。在这里，我们建议采用新兴的冷冻电子显微镜（Cryo-EM）的方法来阐明的AdV核核心的结构组织减去二十面体衣壳区域的颗粒（病毒体）的图像和重建的核心区域（S）独立不强加二十面体对称。在目标1中，我们将重建成熟HAdV 5的核核心组织。此外，我们将阐明重组的核芯热处理后，是已知的模仿发生在部分拆卸和内体逃逸的结构变化。在Aim 2中，我们将对HAdV-C5的超热稳定和非感染性形式（ts 1突变体）中观察到的独特核核心组织进行成像，并阐明ts 1颗粒更高热稳定性的分子基础。总的来说，这些研究提供了腺病毒核蛋白核心组织的结构细节，揭示了基因组包装成腺病毒的机制以及HAdV成熟后发生的变化。此外，这些研究将阐明在HAdV病毒粒子的部分分解和感染过程中发生的变化，通过热处理来模拟。

Human adenoviruses (HAdVs) are large (MW ~150 mDa), complex, non-enveloped dsDNA viruses that cause acute respiratory disease, epidemic keratoconjunctivitis and gastrointestinal infections. Significantly, adenoviruses (AdVs) are being used as viral vectors for gene, vaccine and Oncolytic viral therapies. Although the in situ structures and arrangement of major and minor capsid proteins (CPs) in the assembled (icosahedral) viral capsid have been determined at near-atomic resolution, there is no information currently available on how the nucleoprotein core, comprising linear dsDNA (~36 kbp) and “histone like” core proteins, is organized. The core density shows up as smeared ball in the icosahedral reconstructions of the AdVs. This is in contrast to characteristic spooled organization of the dsDNA seen in bacteriophages and herpesviruses. The details of nuclear core organization provide clues into the mechanism of genome packaging into AdVs, which is still unresolved. Here, we propose to employ emerging cryo-electron microscopy (Cryo-EM) methods to elucidate the structural organization of the AdV nuclear core by subtracting the icosahedral capsid regions from the particle (virion) images and reconstructing the core region(s) independently without imposing icosahedral symmetry. In Aim 1, we will reconstruct the nuclear core organization of mature HAdV5. Additionally, we will elucidate the reorganization of the nuclear core upon heat treatment that is known to mimic the structural changes that occur during partial disassembly and endosome escape. In Aim2 we will image the distinct nuclear core organization observed in hyper-thermostable and noninfectious form (ts1 mutant) of HAdV-C5 as well as elucidate the molecular basis of its greater thermo-stability of ts1 particles. Collectively, these studies provide structural details of nucleoprotein core organization in AdVs with implications for revealing the mechanism of genome packaging into AdVs as well as the changes that occur upon HAdV maturation. In addition, these studies will elucidate the changes that occur during the partial disassembly and infection of HAdV virions, emulated by the heat treatment.