Fig 2: Overexpression of G3BP1 enhances SG assembly and inhibits BPIV3 replication. (A) G3BP1 overexpression promotes the formation of SGs in cells. HeLa cells were transfected with VR-G3BP1-3×FLAG or VR-3×FLAG plasmids (100 ng, 96-well plates) for 36 h and detected with rabbit anti-G3BP1 antibody, which was followed by probing with goat anti-rabbit-coupled Alexa Fluor 488 antibody. Cell nuclei were restained with DAPI. The rate of SG formation was calculated after counting the cells with SG formation and the total number of cells. (B) Overexpression of G3BP1 inhibits the growth of BPIV3. HeLa cells were transfected with VR-G3BP1-3×FLAG or VR-3×FLAG plasmids; controls were not transfected. After infection with BPIV3 (MOI = 2.0), viral titers in cell culture at different time points were determined using the TCID50 method. (C) G3BP1 overexpression reduces the BPIV3 viral copy number. HeLa cells were transfected with VR-G3BP1-3×FLAG or VR-3×FLAG plasmid (500 ng, 24-well plate) for 36 h. The controls were not transfected with the plasmid. Simulated infection with BPIV3 (MOI = 2.0) for 24 h was used to detect intracellular viral mRNA levels using qRT-PCR. GAPDH mRNA expression was used as an internal control. (D) Overexpression of G3BP1 reduces BPIV3-N protein expression. Cells were infected or not infected with BPIV3 after transfection (MOI = 2.0). Proteins were examined using an anti-G3BP1 antibody against cell lysates and an anti-BPIV3-N antibody. Actin was used as an endogenous cellular reference. Data are expressed as mean ± SD, n = 3. The significance of difference between the groups was performed by ANOVA (* stands for p < 0.05; ** stands for p < 0.01; *** stands for p < 0.001).

Fig 3: BPIV3 infection inhibits the formation of SGs induced by SA and downregulates G3BP1 protein expression. (A, B) Cells were either mock-infected or uninfected with BPIV3 (MOI = 2.0) after treatment with AS (0.5 mM) for 45 min at the indicated infection time points prior to fixation. Cells were stained with rabbit anti-G3BP1 antibody and mouse anti-BPIV3-N antibody, and then labeled with goat anti-rabbit coupled Alexa Fluor 594 antibody and goat anti-mouse coupled Alexa Fluor 488 antibody. Cell nuclei were restained with DAPI. (C) BPIV3 infection resulted in a decrease in the percentage of cells forming SGs. The number of cells forming SGs and the total number of cells were counted for AS-treated cells to determine the percentage of cells containing stress granules. (D) BPIV3 infection downregulates G3BP1 mRNA levels in MDBK cells. G3BP1 mRNA expression was measured using qRT-PCR at different time points of infection (3, 6, 9, 12, and 24 h) using primers specific for the BPIV3-M gene. GAPDH mRNA expression was used as an internal control to calculate relative levels of gene expression. (E) BPIV3 infection downregulates G3BP1 protein levels in MDBK cells. Cell lysates were prepared from the same cell culture samples as those used to perform the qRT-PCR assay. G3BP1 expression was examined using protein blotting with anti-G3BP1 and anti-BPIV3-N antibodies. Tubulin was used as an endogenous control. Data are expressed as mean ± SD, n = 3. The significance of difference between the groups was performed by ANOVA (* stands for p < 0.05; ** stands for p < 0.01; *** stands for p < 0.001; ns stands for p > 0.05).

Fig 4: Internal m7G-modified Lrp1 mRNA is shuttled by QKI7 into SGs thus repressing its translation.a BMDMs were administrated with HDM (200 µg/well), and relative Lrp1 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). b In BMDMs from G3bp1f/f and G3bp1mac-/- mice, relative Lrp1 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). c Sequencing read clusters from m7G MeRIP-seq analysis of Lrp1 mRNA in PMs and top consensus motif identified by HOMER with MeRIP-seq peaks. d m7G modification of Lrp1 mRNA in primary macrophages was examined by m7G-RIP-qRT-PCR (n = 3, biological replicates). (two-tailed unpaired Student’s t-test with Welch’s correction). e Immunofluorescence of G3BP1 and m7G-modified mRNA, and RNA-scope imaging of Lrp1 mRNA in PMs. f Immunofluorescence of G3BP1 and QKI7, and RNA-scope imaging of Lrp1 mRNA in PMs. g The association between QKI7 and Lrp1 mRNA in primary macrophages was determined by RIP-qRT-PCR (n = 3, biological replicates). (two-tailed unpaired Student’s t-test). h The association between the KH domain of QKI7 and Lrp1 mRNA in RAW 264.7 cells was determined by RIP-qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). i The association between QKI7 and Lrp1 mRNA in PMs was determined by RNA pull-down assay. j In QKI7-overexpressed RAW 264.7 cells stimulated by HDM (200 µg/well), relative Lrp1 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments. Δ not significant.

Fig 5: SG assembly in macrophages sequesters Lrp1 mRNA to suppress its translation.a Venn diagram indicating overlap of mRNAs with SG-enrichment and increased G3BP1-binding under stress conditions (using the OmicStudio tools at https://www.omicstudio.cn/tool). b The association between G3BP1 and Lrp1 mRNA in primary macrophages was determined by RIP-qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). c The association between the NTF2 domain of G3BP1 and Lrp1 mRNA in RAW 264.7 cells was determined by RIP-qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). d Immunofluorescence of G3BP1 and RNA-scope imaging of Lrp1 mRNA in PMs. e The association between G3BP1 and Lrp1 mRNA in PMs was determined by RNA pull-down assay. f G3BP1 and LRP1 protein levels in primary macrophages stimulated with HDM (100 µg/well) or NaAsO2 (50 µM) were examined by Western blot. g LRP1 protein level on the surface of PMs stimulated with HDM (100 µg/well) or NaAsO2 (50 µM) were examined by flow cytometry (n = 3, biological replicates). (Dunnett’s multiple comparisons test). h G3BP1 and LRP1 protein levels in primary macrophages of G3bp1f/f and G3bp1mac-/- mice stimulated with HDM (100 µg/well) were examined by Western blot. i LRP1 protein level on the surface of BMDMs of G3bp1f/f and G3bp1mac-/- mice stimulated with HDM (100 µg/well) were examined by flow cytometry (n = 3, biological replicates). (Tukey’s HSD). j LRP1 protein level on the surface of PMs of G3bp1f/f and G3bp1mac-/- mice were examined by flow cytometry (n = 3, biological replicates). (two-tailed unpaired Student’s t-test). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.