title: Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis.
abstract: In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503-1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany. griffiths@embl-heidelberg.de
authors.forename: G
authors.initials: G
authors.lastname: Griffiths
authors.forename: R
authors.initials: R
authors.lastname: Wepf
authors.forename: T
authors.initials: T
authors.lastname: Wendt
authors.forename: J K
authors.initials: JK
authors.lastname: Locker
authors.forename: M
authors.initials: M
authors.lastname: Cyrklaff
authors.forename: N
authors.initials: N
authors.lastname: Roos
mesh_terms: D004229:Dithiothreitol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008855:Microscopy, Electron, Scanning; D014616:Vaccinia virus / Q000502:physiology / Q000648:ultrastructure*; D014771:Virion / Q000502:physiology / Q000648:ultrastructure*; D019065:Virus Assembly
chemical_list: D004229:Dithiothreitol