Influenza A virus-induced production of PI4P at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

ABSTRACT: To further understand the molecular mechanisms by which RAB11A is hijacked in IAV-infected cells, we sought to identify viral-induced changes in RAB11A interactome. To this end, we used a TurboID-based proximity labelling approach, most suitable for the detection of weak or transient protein-protein interactions (Figure 2A). TurboID is a highly active biotin ligase that rapidly converts biotin into biotin–AMP, a reactive intermediate which covalently labels proximal proteins within a 10-30 nm radius(REF DOI : 10.1038/s41596-020-0399-0). The RAB11A protein was fused to TurboID and stably expressed in A549 cells by lentiviral transduction (Supplementary Figure 2A western blot). The resulting A549-TurboID-RAB11A cell population was infected with the WSN virus at a MOI of 5 PFU/cell - conditions in which all cells are positive for the viral nucleoprotein (NP) upon immunostaining at 8 hpi (Supplementary Figure 2B IF-NP), or mock-infected. At 8 hpi, biotin was added to the culture medium for 10 min before cell lysates were prepared, and biotinylated proteins were purified with streptavidin beads, as described in REF. A fraction of the total lysates and the biotinylated eluates were analysed by mass spectrometry based-proteomics. Four independent biological replicates were performed.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Line Cell

SUBMITTER: Thibaut Douché  

LAB HEAD: Nadia Naffakh

PROVIDER: PXD053858 | Pride | 2025-06-19

REPOSITORIES: Pride