Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

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.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009975.All other data generated or analysed during this study are included in the manuscript and supporting files.

The following data sets were generated

Article and author information

Author details

  1. Philip V'kovski

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8366-1220
  2. Markus Gerber

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Jenna Kelly

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  4. Stephanie Pfaender

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Nadine Ebert

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Sophie Braga Lagache

    Mass Spectrometry and Proteomics Core Facility, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Cedric Simillion

    Mass Spectrometry and Proteomics Core Facility, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Jasmine Portmann

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  9. Hanspeter Stalder

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  10. Véronique Gaschen

    Division Veterinary Anatomy, University of Bern, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  11. Rémy Bruggmann

    Interfaculty Bioinformatics Unit, University of Bern, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4733-7922
  12. Michael H Stoffel

    Division of Veterinary Anatomy, University of Bern, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4699-5125
  13. Manfred Heller

    Mass Spectrometry and Proteomics Core Facility, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  14. Ronald Dijkman Dijkman

    Institute of Virology and Immunology IVI, Bern, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  15. Volker Thiel

    Institute of Virology and Immunology IVI, Bern, Switzerland
    For correspondence
    volker.thiel@vetsuisse.unibe.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5783-0887

Funding

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (173085)

  • Philip V'kovski
  • Volker Thiel

European Commission (748627)

  • Stephanie Pfaender
  • Volker Thiel

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (160780)

  • Jenna Kelly
  • Nadine Ebert
  • Volker Thiel

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Karla Kirkegaard, Stanford University School of Medicine, United States

Publication history

  1. Received: September 14, 2018
  2. Accepted: January 11, 2019
  3. Accepted Manuscript published: January 11, 2019 (version 1)
  4. Version of Record published: February 12, 2019 (version 2)

Copyright

© 2019, V'kovski et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Philip V'kovski
  2. Markus Gerber
  3. Jenna Kelly
  4. Stephanie Pfaender
  5. Nadine Ebert
  6. Sophie Braga Lagache
  7. Cedric Simillion
  8. Jasmine Portmann
  9. Hanspeter Stalder
  10. Véronique Gaschen
  11. Rémy Bruggmann
  12. Michael H Stoffel
  13. Manfred Heller
  14. Ronald Dijkman Dijkman
  15. Volker Thiel
(2019)
Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling
eLife 8:e42037.
https://doi.org/10.7554/eLife.42037

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