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Proximity labeling and LC-MS/MS based identification of the stress granule microenvironment induced by different viruses

ABSTRACT: To dissect and compare the protein composition of stress granules formed during infections with two different RNA viruses as well as oxidative stress, we developed a recombinant mouse fibroblast cell line stably expressing a fusion protein of the stress granule marker G3BP1 with APEX2 and GFP. When stress granules are induced in these cells, the fusion protein accumulates in the cytoplasmic condensates and allows the biotinylation of the SG proteome based on the APEX2 peroxidase. The proximity labeled proteins can then be affinity purified and identified by quantitative LC-MS/MS. To directly compare different stressed and unstressed conditions, we combined the proximity labeling approach with stable isotope labeling of amino acids in cell culture (SILAC). In the first experiment (Exp 1, 2019 data), we compared the SGs induced by MHV infection with canonical SGs induced by sodium arsenite treatment or the unstressed cells. In a second and third experiment (Exp 2 and 3, 2023 and 2025 data), we expanded our investigations to SGs induced by SFV infection to directly compare the SG proteome between different viruses and at different time points during infection.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Manfred Heller

LAB HEAD: Volker Thiel

PROVIDER: PXD077432 | Pride | 2026-08-07

REPOSITORIES: Pride

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Project description:A total of 432 genes were found to be differentially expressed in M1SF370 bacterial population internalized in Detroit 562 human pharyngeal cells when compared with the same strain incubated in the absence of Detroit 562 cells. While most of them (349/432 i.e. 80.8%) were up regulated, 83 genes were down regulated contributing to 19.2% of the total differentiated genes. The major contributor of the latter category was phage-related genes (35 genes). Almost ¼ of these genes (106) belonged to a category of Unknown or possible predicted function. Most notably, up-regulated genes belonged to amino acid transport , cell division, cell envelope biogenesis, DNA replication correlated well with up-regulated 67 genes belonged to translation and ribosomal structure. Further, up-regulation of 12/15 virulence-related genes indicated that human host cell internalized bacteria are highly virulent as compared to laboratory grown culture in test-tubes. S. pyogenes strain type M1 SF370 (wild-type) was procured from ATCC (ATCC 700294). Detroit 562 pharyngeal cells were obtained from ATCC and maintained in MEM with 10% FBS in humidified CO2-incubator. Purified cDNA preparations from the Detroit cells-internalized bacteria and that were cultured without Detroit cells, were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using four independently isolated RNA preparations (biological replicates), a total of 8 experiments (incorporating 4 dye swaps) were performed. Accordingly eight hybridization measurements for this mutant were obtained. Exp-1 and -2 (GSM687276, GSM687310-dye swap) are the technical replicates of the biological sample-1, Exp-3 and -4 (GSM687311, GSM687312-dye swap) are technical replicates of biological sample-2, Exp-5 and -6, (GSM687313,GSM687319-dye swap,) are technical replicates of the biological sample-3, and finally Exp-7 and -8, (GSM687320,GSM687321-dye swap) are technical replicates of the biological sample-4. .

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	20190114_10_SS_i01.raw	Raw
	20190114_10_SS_i02.raw	Raw
	20190114_10_SS_i03.raw	Raw
	20190114_10_SS_i04.raw	Raw
	20190114_1_SS_i01.raw	Raw

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Proximity labeling and LC-MS/MS based identification of the stress granule microenvironment induced by different viruses

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