skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Visualization of the herpes simplex virus portal in situ by cryo-electron tomography

Abstract

Herpes simplex virus type 1 (HSV-1), the prototypical herpesvirus, has an icosahedral nucleocapsid surrounded by a proteinaceous tegument and a lipoprotein envelope. As in tailed bacteriophages, the icosahedral symmetry of the capsid is broken at one of the 12 vertices, which is occupied by a dodecameric ring of portal protein, UL6, instead of a pentamer of the capsid protein, UL19. The portal ring serves as a conduit for DNA entering and exiting the capsid. From a cryo-EM reconstruction of capsids immuno-gold-labeled with anti-UL6 antibodies, we confirmed that UL6 resides at a vertex. To visualize the portal in the context of the assembled capsid, we used cryo-electron tomography to determine the three-dimensional structures of individual A-capsids (empty, mature capsids). The similarity in size and overall shape of the portal and a UL19 pentamer - both are cylinders of {approx} 800 kDa - combined with residual noise in the tomograms, prevented us from identifying the portal vertices directly; however, this was accomplished by a computational classification procedure. Averaging the portal-containing subtomograms produced a structure that tallies with the isolated portal, as previously reconstructed by cryo-EM. The portal is mounted on the outer surface of the capsid floor layer, with its narrow endmore » pointing outwards. This disposition differs from that of known phage portals in that the bulk of its mass lies outside, not inside, the floor. This distinction may be indicative of divergence at the level of portal-related functions other than its role as a DNA channel.« less

Authors:
 [1];  [1];  [1];  [2];  [1];  [3];  [4];  [4];  [5]
  1. Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Building 50, Rm 1517, MSC 8025, 50 South Drive, National Institutes of Health, Bethesda, MD 20892-8025 (United States)
  2. (United States)
  3. Department of Cell Biology, Washington University School of Medicine, St. Louis, MO 63110 (United States)
  4. Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, VA 22908 (United States)
  5. Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Building 50, Rm 1517, MSC 8025, 50 South Drive, National Institutes of Health, Bethesda, MD 20892-8025 (United States). E-mail: Alasdair_Steven@nih.gov
Publication Date:
OSTI Identifier:
20977021
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 361; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2006.10.047; PII: S0042-6822(06)00804-X; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; BACTERIOPHAGES; DNA; ELECTRON MICROSCOPY; HERPES SIMPLEX; LIPOPROTEINS; TOMOGRAPHY

Citation Formats

Cardone, Giovanni, Winkler, Dennis C., Trus, Benes L., Imaging Sciences Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, Cheng, Naiqian, Heuser, John E., Newcomb, William W., Brown, Jay C., and Steven, Alasdair C. Visualization of the herpes simplex virus portal in situ by cryo-electron tomography. United States: N. p., 2007. Web. doi:10.1016/j.virol.2006.10.047.
Cardone, Giovanni, Winkler, Dennis C., Trus, Benes L., Imaging Sciences Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, Cheng, Naiqian, Heuser, John E., Newcomb, William W., Brown, Jay C., & Steven, Alasdair C. Visualization of the herpes simplex virus portal in situ by cryo-electron tomography. United States. doi:10.1016/j.virol.2006.10.047.
Cardone, Giovanni, Winkler, Dennis C., Trus, Benes L., Imaging Sciences Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, Cheng, Naiqian, Heuser, John E., Newcomb, William W., Brown, Jay C., and Steven, Alasdair C. Thu . "Visualization of the herpes simplex virus portal in situ by cryo-electron tomography". United States. doi:10.1016/j.virol.2006.10.047.
@article{osti_20977021,
title = {Visualization of the herpes simplex virus portal in situ by cryo-electron tomography},
author = {Cardone, Giovanni and Winkler, Dennis C. and Trus, Benes L. and Imaging Sciences Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892 and Cheng, Naiqian and Heuser, John E. and Newcomb, William W. and Brown, Jay C. and Steven, Alasdair C.},
abstractNote = {Herpes simplex virus type 1 (HSV-1), the prototypical herpesvirus, has an icosahedral nucleocapsid surrounded by a proteinaceous tegument and a lipoprotein envelope. As in tailed bacteriophages, the icosahedral symmetry of the capsid is broken at one of the 12 vertices, which is occupied by a dodecameric ring of portal protein, UL6, instead of a pentamer of the capsid protein, UL19. The portal ring serves as a conduit for DNA entering and exiting the capsid. From a cryo-EM reconstruction of capsids immuno-gold-labeled with anti-UL6 antibodies, we confirmed that UL6 resides at a vertex. To visualize the portal in the context of the assembled capsid, we used cryo-electron tomography to determine the three-dimensional structures of individual A-capsids (empty, mature capsids). The similarity in size and overall shape of the portal and a UL19 pentamer - both are cylinders of {approx} 800 kDa - combined with residual noise in the tomograms, prevented us from identifying the portal vertices directly; however, this was accomplished by a computational classification procedure. Averaging the portal-containing subtomograms produced a structure that tallies with the isolated portal, as previously reconstructed by cryo-EM. The portal is mounted on the outer surface of the capsid floor layer, with its narrow end pointing outwards. This disposition differs from that of known phage portals in that the bulk of its mass lies outside, not inside, the floor. This distinction may be indicative of divergence at the level of portal-related functions other than its role as a DNA channel.},
doi = {10.1016/j.virol.2006.10.047},
journal = {Virology},
number = 2,
volume = 361,
place = {United States},
year = {Thu May 10 00:00:00 EDT 2007},
month = {Thu May 10 00:00:00 EDT 2007}
}
  • The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infectedmore » with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.« less
  • We identify an NLS within herpes simplex virus scaffold proteins that is required for optimal nuclear import of these proteins into infected or uninfected nuclei, and is sufficient to mediate nuclear import of GFP. A virus lacking this NLS replicated to titers reduced by 1000-fold, but was able to make capsids containing both scaffold and portal proteins suggesting that other functions can complement the NLS in infected cells. We also show that Vp22a, the major scaffold protein, is sufficient to mediate the incorporation of portal protein into capsids, whereas proper portal immunoreactivity in the capsid requires the larger scaffold proteinmore » pU{sub L}26. Finally, capsid angularization in infected cells did not require the HSV-1 protease unless full length pU{sub L}26 was expressed. These data suggest that the HSV-1 portal undergoes conformational changes during capsid maturation, and reveal that full length pU{sub L}26 is required for this conformational change.« less
  • Cells infected with wild type HSV-1 showed significant lamin A/C and lamin B rearrangement, while U{sub L}34-null virus-infected cells exhibited few changes in lamin localization, indicating that U{sub L}34 is necessary for lamin disruption. During HSV infection, U{sub S}3 limited the development of disruptions in the lamina, since cells infected with a U{sub S}3-null virus developed large perforations in the lamin layer. U{sub S}3 regulation of lamin disruption does not correlate with the induction of apoptosis. Expression of either U{sub L}34 or U{sub S}3 proteins alone disrupted lamin A/C and lamin B localization. Expression of U{sub L}34 and U{sub S}3more » together had little effect on lamin A/C localization, suggesting a regulatory interaction between the two proteins. The data presented in this paper argue for crucial roles for both U{sub L}34 and U{sub S}3 in regulating the state of the nuclear lamina during viral infection.« less
  • A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX;more » gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.« less
  • Bacteriophage P1 has a contractile tail that targets the conserved lipopolysaccharide on the outer membrane surface of the host for initial adsorption. The mechanism by which P1 DNA enters the host cell is not well understood, mainly because the transient molecular interactions between bacteriophage and bacteria have been difficult to study by conventional approaches. Here, we engineered tiny E. coli host cells so that the initial stages of P1-host interactions could be captured in unprecedented detail by cryo-electron tomography. Analysis of three-dimensional reconstructions of frozen-hydrated specimens revealed three predominant configurations: an extended tail stage with DNA present in the phagemore » head, a contracted tail stage with DNA, and a contracted tail stage without DNA. Comparative analysis of various conformations indicated that there is uniform penetration of the inner tail tube into the E. coli periplasm and a significant movement of the baseplate away from the outer membrane during tail contraction.« less