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Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling

Philip V'kovski, Markus Gerber, Jenna Kelly, Stephanie Pfaender, Nadine Ebert, Sophie Braga Lagache, Cedric Simillion, Jasmine Portmann, Hanspeter Stalder, Veronique Gaschen, Remy Bruggmann, Michael Stoffel, Manfred Heller, View ORCID ProfileRonald Dijkman, Volker Thiel
doi: https://doi.org/10.1101/417907
Philip V'kovski
Institute for Virology and Immunology;
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Markus Gerber
Institute for Virology and Immunology;
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Jenna Kelly
Institute for Virology and Immunology;
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Stephanie Pfaender
Institute for Virology and Immunology;
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Nadine Ebert
Institute for Virology and Immunology;
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Sophie Braga Lagache
Mass Spectrometry & Proteomics Core Facility, Dept for BioMedical Research, University of Bern;
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Cedric Simillion
Mass Spectrometry & Proteomics Core Facility, Dept for BioMedical Research, University of Bern;
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Jasmine Portmann
Institute for Virology and Immunology;
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Hanspeter Stalder
Institute for Virology and Immunology;
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Veronique Gaschen
Division Veterinary Anatomy, Vetsuisse Faculty, University of Bern, Bern, Switzerland;
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Remy Bruggmann
Interfaculty Bioinformatics Unit and SIB Swiss Institute of Bioinformatics, University of Bern
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Michael Stoffel
Division Veterinary Anatomy, Vetsuisse Faculty, University of Bern, Bern, Switzerland;
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Manfred Heller
Mass Spectrometry & Proteomics Core Facility, Dept for BioMedical Research, University of Bern;
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Ronald Dijkman
Institute for Virology and Immunology;
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  • ORCID record for Ronald Dijkman
Volker Thiel
Institute for Virology and Immunology;
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  • For correspondence: volker.thiel@vetsuisse.unibe.ch
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Abstract

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 14, 2018.
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Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling
Philip V'kovski, Markus Gerber, Jenna Kelly, Stephanie Pfaender, Nadine Ebert, Sophie Braga Lagache, Cedric Simillion, Jasmine Portmann, Hanspeter Stalder, Veronique Gaschen, Remy Bruggmann, Michael Stoffel, Manfred Heller, Ronald Dijkman, Volker Thiel
bioRxiv 417907; doi: https://doi.org/10.1101/417907
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Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling
Philip V'kovski, Markus Gerber, Jenna Kelly, Stephanie Pfaender, Nadine Ebert, Sophie Braga Lagache, Cedric Simillion, Jasmine Portmann, Hanspeter Stalder, Veronique Gaschen, Remy Bruggmann, Michael Stoffel, Manfred Heller, Ronald Dijkman, Volker Thiel
bioRxiv 417907; doi: https://doi.org/10.1101/417907

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