Proximity biotinylation and LC-MSMS identification of the molecular microenvironment of coronavirus replication complexes

ABSTRACT: To investigate virus-host interactions at the site of coronavirus replication, we developed a biotin-based proximity labelling approach by engineering a promiscuous biotin ligase, BirA-R118G, within the coronavirus replication and transcription complex (RTC). BirA-R118G was fused to non-structural protein 2 to generate MHV- BirA-R118G-nsp2. During the course of infection, viral and host factors in proximity of the viral RTC become covalently biotinylated, allowing affinity purification of biotin-tagged factors and mass spectrometry identification of RTC-proximal viral and host factors. Results provide a comprehensive library of RTC-associated factors which likely include crucial factors that promote, orchestrate and assist viral replication within the viral RTC microenvironment.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Murine Hepatitis Virus Strain A59 Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Manfred Heller  

LAB HEAD: Volker Thiel

PROVIDER: PXD009975 | Pride | 2019-01-16

REPOSITORIES: Pride