Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

Abstract

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction.

Architecture of Thylakoid Membrane Networks

“…The structure of many cellular objects that had previously been observed by means of conventional EM has been reinterpreted, new structural motifs have been found, and prevailing paradigms are being challenged. Applied to large molecular assemblies, EMT has provided invaluable insights into the structure of the ribosome (Zhao et al, 2004a, b), proteosome (Böhm et al, 2000;Medalia et al, 2002), flagellar motors (Nicastro et al, 2005;Murphy et al, 2006), nuclear pore complexes (Stoffler et al, 2003;Beck et al, 2007) and viruses Cyrklaff et al, 2007;Subramaniam et al, 2007;Dai et al, 2008). It has likewise been instrumental in revealing the intricate organization of complex, pleiomorphic structures, which impede high resolution 3D analysis by other methods, such as the cytoskeleton, in eukaryotes (Kurner et al, 2004;Hoog and Antony, 2007;O'Toole et al, 2007;Koning et al, 2008;Perkins et al, 2008) and in prokaryotes (Scheffel et al, 2006;Jensen and Briegel, 2007), and the spindle pole body of yeast (O'Toole et al, 1999;Melloy et al, 2007).…”

Cryo-electron Tomography of Whole Cells

“…1a) recorded and computed in the same conditions as in the whole cell approach [1]. We surprisingly found that the major changes to the vaccinia structure happen right before the internalization into the host cell cytoplasm [5]. This is the stage when the virus interacts with receptors exposed on the host cell plasma membrane.…”

Cellular structural biology as revealed by cryo-electron tomography

“…This generally limits the type of specimens that can be imaged by EM to isolated viruses (Hong et al, 2015;Murata et al, 2010;Dai et al, 2010), protein complexes (Koyfman et al, 2011;Nickell et al, 2007a,b), organelles (Eibauer et al, 2015;Nicastro et al, 2000;Beck et al, 2007Beck et al, , 2004, smaller prokaryotic cells (Briegel et al, 2015;Zhao et al, 2014) and thin peripheral regions of eukaryotic cells (Hu, 2014;Ben-Harush et al, 2010;Patla et al, 2010;Cyrklaff et al, 2007). Cells up to 1 μm in thickness can be imaged by EM, as shown for Ostreococcus tauri, the smallest eukaryotic cell (Henderson et al, 2007); this has provided a unique insight into the ultrastructure and characteristics of the cells during different stages of their cell cycle.…”