The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

ABSTRACT All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an i...

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Autores principales:	Juan Fontana, Doina Atanasiu, Wan Ting Saw, John R. Gallagher, Reagan G. Cox, J. Charles Whitbeck, Lauren M. Brown, Roselyn J. Eisenberg, Gary H. Cohen
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	cryo-electron microscopy cryo-electron tomography HSV subtomogram averaging gB herpesviruses Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bedf0d56e13a4379a5a712758525c86a
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spelling	oai:doaj.org-article:bedf0d56e13a4379a5a712758525c86a2021-11-15T15:51:44ZThe Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane10.1128/mBio.01268-172150-7511https://doaj.org/article/bedf0d56e13a4379a5a712758525c86a2017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01268-17https://doaj.org/toc/2150-7511ABSTRACT All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity. IMPORTANCE The herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope with the host membrane to start an infection. This process is mediated by a viral surface protein that transitions from an initial conformation (prefusion) to a final, more stable, conformation (postfusion). However, the prefusion conformation of the herpesvirus fusion protein (gB) is poorly understood. To elucidate the structure of the prefusion conformation of HSV type 1 gB, we have employed cryo-electron microscopy to study gB molecules expressed on the surface of vesicles. Using different approaches to label gB’s domains allowed us to model the structures of the prefusion and intermediate conformations of gB. Overall, our findings enhance our understanding of HSV fusion and lay the groundwork for the development of new ways to prevent and block HSV infection.Juan FontanaDoina AtanasiuWan Ting SawJohn R. GallagherReagan G. CoxJ. Charles WhitbeckLauren M. BrownRoselyn J. EisenbergGary H. CohenAmerican Society for Microbiologyarticlecryo-electron microscopycryo-electron tomographyHSVsubtomogram averaginggBherpesvirusesMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	cryo-electron microscopy cryo-electron tomography HSV subtomogram averaging gB herpesviruses Microbiology QR1-502
spellingShingle	cryo-electron microscopy cryo-electron tomography HSV subtomogram averaging gB herpesviruses Microbiology QR1-502 Juan Fontana Doina Atanasiu Wan Ting Saw John R. Gallagher Reagan G. Cox J. Charles Whitbeck Lauren M. Brown Roselyn J. Eisenberg Gary H. Cohen The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane
description	ABSTRACT All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity. IMPORTANCE The herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope with the host membrane to start an infection. This process is mediated by a viral surface protein that transitions from an initial conformation (prefusion) to a final, more stable, conformation (postfusion). However, the prefusion conformation of the herpesvirus fusion protein (gB) is poorly understood. To elucidate the structure of the prefusion conformation of HSV type 1 gB, we have employed cryo-electron microscopy to study gB molecules expressed on the surface of vesicles. Using different approaches to label gB’s domains allowed us to model the structures of the prefusion and intermediate conformations of gB. Overall, our findings enhance our understanding of HSV fusion and lay the groundwork for the development of new ways to prevent and block HSV infection.
format	article
author	Juan Fontana Doina Atanasiu Wan Ting Saw John R. Gallagher Reagan G. Cox J. Charles Whitbeck Lauren M. Brown Roselyn J. Eisenberg Gary H. Cohen
author_facet	Juan Fontana Doina Atanasiu Wan Ting Saw John R. Gallagher Reagan G. Cox J. Charles Whitbeck Lauren M. Brown Roselyn J. Eisenberg Gary H. Cohen
author_sort	Juan Fontana
title	The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane
title_short	The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane
title_full	The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane
title_fullStr	The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane
title_full_unstemmed	The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane
title_sort	fusion loops of the initial prefusion conformation of herpes simplex virus 1 fusion protein point toward the membrane
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/bedf0d56e13a4379a5a712758525c86a
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The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

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