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

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

eLife (2019-01-12)
Philip V'kovski, Markus Gerber, Jenna Kelly, Stephanie Pfaender, Nadine Ebert, Sophie Braga Lagache, Cedric Simillion, Jasmine Portmann, Hanspeter Stalder, Véronique Gaschen, Rémy Bruggmann, Michael H Stoffel, Manfred Heller, Ronald Dijkman, Volker Thiel
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-β-Actin−Peroxidase antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
Sigma-Aldrich
Biotin, ≥99% (HPLC), lyophilized powder
Sigma-Aldrich
Anti-Puromycin Antibody, clone 12D10, clone 12D10, from mouse