Dataset Information

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

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			Action	DRS
	20211213-Homo_sapiens.fasta	Fasta
	20220723_Q1_TD_ColQ1-123_Brault_IP-F-1.raw	Raw
	20220723_Q1_TD_ColQ1-123_Brault_IP-F-2.raw	Raw
	20220723_Q1_TD_ColQ1-123_Brault_IP-F-3.raw	Raw
	20220723_Q1_TD_ColQ1-123_Brault_IP-F-4.raw	Raw

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Influenza A virus induces PI4P production at the endoplasmic reticulum in an ATG16L1-dependent manner to promote the egress of viral ribonucleoproteins.

Alemany Carla C Da Graça Juliane J Giai Gianetto Quentin Q Dupont Maud M Paisant Sylvain S Douché Thibaut T Isel Catherine C Delevoye Cédric C Danglot Lydia L Matondo Mariette M Morel Etienne E Brault Jean-Baptiste JB Naffakh Nadia N

PLoS biology 20250716 7

The genomic RNAs of influenza A viruses (IAVs) are replicated in the nucleus of infected cells in the form of viral ribonucleoproteins (vRNPs) before being exported to the cytoplasm. The small GTPase RAB11A is involved in the transport of vRNPs to the sites of viral assembly at the plasma membrane, but the molecular mechanisms involved remain largely unknown. Here we show that IAV infection remodels the architecture of the endoplasmic reticulum (ER) sheets, where vRNPs tend to accumulate in the ...[more]

PMID: 40668844

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Project description:In this study, we used the in vivo proximity labeling biotin identification (BioID; Roux et al., 2012) method to capture proteins of the microenvironment of the greenbeard determinant of communication (DOC) proteins SmDOC1 and SmDOC2 in the filamentous fungus Sordaria macrospora. The genes Smdoc1 (SMAC_06903) and Smdoc2 (SMAC_06902) were fused to the TurboID ligase via a GGGGS x 2 Linker (Branon et al., 2018) and integrated at their native chromosomal locus. To ensure authentic expression regulation, the Smdoc-TurboID fusion genes were under the control of their native 5’ regions. The homologous doc genes of the closely related fungus Neurospora crassa were shown to be involved in long distance kind discrimination, regulation hyphal fusion upon compatible allorecognition (Heller et al., 2016). The S. macrospora Smdoc1/2 are required for proper sexual development. Our SmDOC1-BioID experimental setup comprises five biological replicates of the strain expressing the Smdoc1-TurboID fusion gene from its native locus and a control strain expressing an egfp-TurboID fusion gene under control of the 1 kb 5’ Smdoc1 promoter region, which was ectopically integrated into the S. macrospora wild type. Our SmDOC2-BioID experimental setup consists of four biological replicates of three strains: 1) a strain expressing the free egfp-TurboID fusion gene under control of the 1 kb 5’ Smdoc1 promoter region, ectopically integrated into the S. macrospora wild type; 2) a strain expressing the Smdoc2-TurboID fusion gene from its native locus; 3) a strain expressing the Smdoc2-TurboID fusion gene from its native locus in the Δmak2 deletion strain background which lacks the MAP kinase mak2 of the pheromone response pathway (Schmidt et al., 2020). To account for possible expression artefacts among strains used in the SmDOC1- and SmDOC2-BioID experiments, we performed additional input controls of the crude protein extract prior to biotin affinity purification. Roux, K. J., Kim, D. in, Raida, M., & Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196(6), 801–810. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology, 36(9), 880–887. Heller, J., Zhao, J., Rosenfield, G., Kowbel, D. J., Gladieux, P., & Glass, N. L. (2016). Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote. PLOS Biology, 14(4), e1002431. Schmidt, S., Märker, R., Ramšak, B., Beier-Rosberger, A. M., Teichert, I., & Kück, U. (2020). Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes. Frontiers in Microbiology, 11, 1722.