SARS-CoV-2 Envelope TurboID LFQ using internal tagging

ABSTRACT: SARS-CoV-2 is the causative agent of the Covid-19 pandemic. Here we investigated the cell biology of the SARS-CoV-2 protein Envelope (E). Envelope is a transmembrane protein that oligomerises to form a pentameric cation channel and is essential for high viral titres. One of SARS-CoV-2's four structural proteins (along with Spike (S), Membrane (M), and Nucleocapsid (N)), Envelope is present in the virion membrane. However, most Envelope is not incorporated into the virion, suggesting it has additional functions in disrupting host cell biology. The study by Pearson et al. investigated the trafficking motifs encoded and the host proteins by Envelope to achieve its trafficking itinerary, with the major focus being how Envelope traffics to lysosomes to cause their deacidification. In this study, Pearson et al. investigated the proximal proteome of Envelope by use of TurboID proximity biotinylation proteomics with label-free quantification (LFQ). Envelope was tagged internally which differs from previous approaches which inadvertently disrupt essential trafficking motifs via the position of the affinity or biotinylation tag. The proteomics was performed using Envelope tagged at three different positions (Site3 - 'TAL', Site 4 - 'VNVS', Extreme C-terminus - 'Cterm') and used both wildtype Envelope and Envelope mutants of interest identified through our cell biology studies of key Envelope trafficking motifs ('Delta' - deletion of C-terminal DLLV; DEWV and SVKI - replacement of C-terminal DLLV with indicated amino acids; 'AxA' - R61A and K63A mutations (not included in the final manuscript)). The dataset also includes TurboID alone ('Cyto') as a control. Each condition was assayed in triplicate. These data identified many novel potential interactors of Envelope, some of which have been confirmed by immunoprecipitation biochemical experiments.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Epithelial Cell, Cell Culture

DISEASE(S): Severe Acute Respiratory Syndrome

SUBMITTER: Guy Pearson  

LAB HEAD: Jeremy Carlton

PROVIDER: PXD045299 | Pride | 2024-02-27

REPOSITORIES: Pride