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

Philip V'Kovski,Hanspeter Stalder,Nadine Ebert,Volker Thiel,Véronique Gaschen,Cedric Simillion,Jenna Kelly,Sophie Braga Lagache,Jasmine Portmann,Manfred Heller,Stephanie Pfaender,Ronald Dijkman,Markus Gerber,Michael H Stoffel,Rémy Bruggmann

doi:10.7554/elife.42037

Philip V'Kovski, Hanspeter Stalder + Show 13 more

Open Access

https://doi.org/10.7554/elife.42037

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Journal: eLife	Publication Date: Jan 11, 2019
Citations: 243	License type: CC BY 4.0

Affiliation: University of Bern, SIB Swiss Institute of Bioinformatics

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.

Highlights

Positive-strand RNA viruses replicate at membranous structures that accommodate the viral replication complex and facilitate RNA synthesis in the cytosol of infected host cells (Romero-Brey and Bartenschlager, 2016; Romero-Brey et al, 2012; Cortese et al, 2017; Knoops et al, 2008; Miorin et al, 2013)
This strategy was chosen because it was recently employed by Freeman et al for a fusion of green fluorescent protein (GFP) with nsp2 and represents the only known site tolerating large insertions within the MHV replicase polyprotein (Freeman et al, 2014)
MHV-GFP-nsp2, which was constructed in parallel and contained the coding sequence of EGFP (Freeman et al, 2014) instead of BirAR118G, was used as a control and reached wild-type virus peak titers, with slightly reduced viral titers at 9 hr post- infection (h.p.i.) compared to MHV-A59 and MHV-BirAR118Gnsp2 (Figure 1b)

Summary

Introduction

Positive-strand RNA viruses replicate at membranous structures that accommodate the viral replication complex and facilitate RNA synthesis in the cytosol of infected host cells (Romero-Brey and Bartenschlager, 2016; Romero-Brey et al, 2012; Cortese et al, 2017; Knoops et al, 2008; Miorin et al, 2013). Microbiology and Infectious Disease eLife digest Coronaviruses can infect the nose and throat and are a main cause of the common cold. In 2002 and 2012, two dangerous new coronaviruses emerged and caused diseases known as SARS and MERS. These viruses caused much more serious symptoms and in some cases proved deadly. To understand why some coronaviruses cause a cold and others kill, they need to learn how people react to virus infection

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