Structural characterization of nucleoprotein cores of human adenoviruses

人腺病毒是一种大分子(MW~150 mda)、复杂、无包膜的双链DNA病毒，可引起急性呼吸道疾病、流行性角结膜炎和胃肠道感染。值得注意的是，腺病毒(ADV)正被用作基因、疫苗和溶瘤病毒治疗的病毒载体。虽然在近原子分辨率下已经确定了组装的(二十面体)病毒衣壳中主要和次要衣壳蛋白(CP)的原位结构和排列，但目前还没有关于核蛋白核心是如何组织的信息，核蛋白核心由线形dsDNA(~36KBP)和类似组蛋白的核心蛋白组成。在ADV的二十面体重建中，核心密度表现为涂抹的球状。这与噬菌体和疱疹病毒中所见的dsDNA特有的假脱氧核糖核酸组织形成了鲜明对比。核核心组织的细节为基因组包装成ADV的机制提供了线索，这一机制仍未解决。在这里，我们建议使用新兴的冷冻电子显微镜(CRYO-EM)方法来阐明ADV核芯的结构组织，方法是从粒子(病毒)图像中减去二十面体衣壳区域，并在不强加二十面体对称性的情况下独立地重建核心区(S)。在目标1中，我们将重建成熟的HAdV5的核核心组织。此外，我们将阐明核芯在热处理时的重组，这是已知的模仿部分拆解和内吞体逸出过程中发生的结构变化。在AIM2中，我们将成像在HAdV-C5的超热和非传染性形式(Ts1突变体)中观察到的独特的核核心组织，并阐明其更大的Ts1颗粒热稳定性的分子基础。总之，这些研究提供了ADV中核蛋白核心组织的结构细节，对于揭示基因组包装到ADV中的机制以及HAdV成熟后发生的变化具有重要意义。此外，这些研究还将阐明热处理模拟的HAdV病毒粒子在部分分解和感染过程中发生的变化。

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.