Extensive Mutagenesis of the HSV-1 gB Ectodomain Reveals Remarkable Stability of Its Postfusion Form

Extensive Mutagenesis of the HSV-1 gB Ectodomain Reveals Remarkable Stability of Its Postfusion Form Viral fusogens mediate the merger of the viral envelope and cellular membrane during viral entry. These proteins share little sequence similarity but all are thought to act by refolding through a series of conformational intermediates from the metastable prefusion form to the stable postfusion form. Crystal structures of both prefusion and postfusion forms have illuminated the conformational pathways of several viral fusogens. By contrast, only the structure of the postfusion form is available for glycoprotein B (gB), the conserved fusogen of herpesviruses. To gain insight into the nature of the fusogenic conformational changes in gB, we used several approaches aimed at engineering the prefusion form of the herpes simplex virus type 1 gB ectodomain, including modifications intended to stabilize the prefusion form and novel mutations aimed at destabilizing the postfusion form. We found that the postfusion conformation of gB is remarkably stable and resistant to perturbations. Several mutations successfully destabilized the gB trimer, identifying regions that are critical for the stability of the postfusion form. Yet, none of the constructs adopted the prefusion conformation. We propose that the soluble ectodomain of gB folds into the postfusion form without first adopting the prefusion intermediate. These results suggest that other regions of gB, including the transmembrane region and the cytoplasmic domain, may be necessary to establish and maintain the metastable prefusion conformation. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

Extensive Mutagenesis of the HSV-1 gB Ectodomain Reveals Remarkable Stability of Its Postfusion Form

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Publisher
Elsevier
Copyright
Copyright © 2013 Elsevier Ltd
ISSN
0022-2836
D.O.I.
10.1016/j.jmb.2013.03.001
Publisher site
See Article on Publisher Site

Abstract

Viral fusogens mediate the merger of the viral envelope and cellular membrane during viral entry. These proteins share little sequence similarity but all are thought to act by refolding through a series of conformational intermediates from the metastable prefusion form to the stable postfusion form. Crystal structures of both prefusion and postfusion forms have illuminated the conformational pathways of several viral fusogens. By contrast, only the structure of the postfusion form is available for glycoprotein B (gB), the conserved fusogen of herpesviruses. To gain insight into the nature of the fusogenic conformational changes in gB, we used several approaches aimed at engineering the prefusion form of the herpes simplex virus type 1 gB ectodomain, including modifications intended to stabilize the prefusion form and novel mutations aimed at destabilizing the postfusion form. We found that the postfusion conformation of gB is remarkably stable and resistant to perturbations. Several mutations successfully destabilized the gB trimer, identifying regions that are critical for the stability of the postfusion form. Yet, none of the constructs adopted the prefusion conformation. We propose that the soluble ectodomain of gB folds into the postfusion form without first adopting the prefusion intermediate. These results suggest that other regions of gB, including the transmembrane region and the cytoplasmic domain, may be necessary to establish and maintain the metastable prefusion conformation.

Journal

Journal of Molecular BiologyElsevier

Published: Jun 12, 2013

References

  • Herpes simplex virus: receptors and ligands for cell entry
    Spear, P.G.
  • Stabilization of short collagen-like triple helices by protein engineering
    Frank, S.; Kammerer, R.A.; Mechling, D.; Schulthess, T.; Landwehr, R.; Bann, J.
  • Entry of herpesviruses into mammalian cells
    Heldwein, E.E.; Krummenacher, C.
  • PHENIX: building new software for automated crystallographic structure determination
    Adams, P.D.; Grosse-Kunstleve, R.W.; Hung, L.W.; Ioerger, T.R.; McCoy, A.J.; Moriarty, N.W.
  • Coot: model-building tools for molecular graphics
    Emsley, P.; Cowtan, K.

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