Abstract
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.
Highlights
Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster [1]
We have recently identified antiviral PML nuclear cages that sequester VZV nucleocapsids and inhibit formation of infectious particles
We determined the 3D distribution of several thousand nucleocapsids within reconstructed volumes of single host cell nuclei and in PML cages as well as their sequestration efficiency and sequestration capacity: more than 98% of nucleocapsids were entrapped within PML cages and individual PML cages could sequester nearly 3,000 nucleocapsids which were cross-linked by an irregular electron-dense meshwork within the PML cages
Summary
Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles) [1]. VZV pathogenesis can be investigated in vivo using xenografts of human dorsal root ganglia (DRG) and skin in a severe combined immunodeficiency (SCID) mouse model [3,4]. Since VZV infectious particles are highly cell-associated, VZV spreads from cell to cell, accompanied by extensive cell-cell fusion and syncytia formation in vitro and polykaryocyte formation in DRG and skin in vivo [5,6,7]. During VZV infection, genome copies are synthesized in nuclear replication compartments and genomic DNA is packaged into icosahedral nucleocapsids formed by ORF40, the major capsid protein, and smaller capsid surface proteins, such as ORF23 protein. Nucleocapsids egress across the nuclear membrane for secondary envelopment in the cytoplasm and are released as enveloped infectious virus particles [1,8]
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