Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America.
PLoS Pathog. 2012;8(6):e1002740. doi: 10.1371/journal.ppat.1002740. Epub 2012 Jun 7.
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.
水痘带状疱疹病毒（VZV）是一种人类α疱疹病毒，可引起水痘（水痘）和带状疱疹（带状疱疹）。与所有疱疹病毒一样，VZV DNA 基因组在核内复制，并包装成核衣壳，这些核衣壳必须穿过核膜才能进入细胞质中的病毒颗粒。我们最近的工作表明，VZV 核衣壳在体外和体内人背根神经节和皮肤异种移植物中被隔离在由早幼粒细胞白血病蛋白（PML）形成的核笼中。我们寻求一种方法来确定疱疹病毒感染细胞核中核衣壳的三维（3D）分布，以及这些独特的 PML 亚核域的 3D 形状、体积和超微结构。在这里，我们报告了一种新的 3D 成像和重建策略的开发，我们称之为连续切片阵列扫描电子显微镜（SSA-SEM）及其在 VZV 感染细胞和这些核 PML 笼中的应用。我们表明，SSA-SEM 允许在足以定位、计数和区分不同类型的 VZV 核衣壳并可视化完整的 PML 笼的分辨率下进行大容量成像和 3D 重建。该方法允许定量确定可以隔离在单个 PML 笼中的核衣壳数量（隔离能力），可以隔离在单个核中的核衣壳的比例（隔离效率），并揭示 PML 笼的超微结构细节。在重建核体积中，超过 98%的所有核衣壳都包含在 PML 笼中，单个 PML 笼可以隔离多达 2780 个核衣壳，电子断层扫描显示这些核衣壳嵌入并交联在这些独特的亚核域内的丝状电子致密网格中。这种 SSA-SEM 分析扩展了我们最近对 PML 笼的表征，并为在单细胞水平上研究病毒组装过程中的事件提供了这种新策略的概念验证。
