Beta-coronaviruses exploit cellular stress responses by modulating TFEB and TFE3 activity

Abstract

Beta-coronaviruses have emerged as a severe threat to global health. Undercovering the interplay between host and beta-coronaviruses is essential for understanding disease pathogenesis and developing efficient treatments. Here we report that the transcription factors TFEB and TFE3 translocate from the cytosol to the nucleus in response to beta-coronavirus infection by a mechanism that requires activation of calcineurin phosphatase. In the nucleus, TFEB and TFE3 bind to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators is significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 is sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 results in increased cellular survival of infected cells but also in reduced lysosomal exocytosis and decreased viral infectivity. These results suggest a central role of TFEB and TFE3 in cellular response to beta-coronavirus infection.

Keywords: Molecular biology; Virology.

Graphical abstract

Figure 1

The transcription factors TFEB and TFE3 translocate to the nucleus in response to MHV infection (A) Immunoblot analysis of protein lysates from HeLa-mCC1a cells treated with DMSO (0 h), infected with MHV for 8 h, 11 h, and 14 h, or incubated in EBSS medium for 4 h (STV). Immunoblots are representative of at least three independent experiments. (B) Representative immunofluorescence images of MHV (M_j1.3) (red) and endogenous TFEB (green). Cells were stained with DAPI (blue). n = 3 independent experiments. Scale bars, 20 μm. (C) Representative immunofluorescence images of HeLa-mCC1a cells treated with DMSO (0 h) or infected with MHV for 16 h and 24 h. Endogenous TFEB and TFE3 are shown in green, MHV M protein in red, and DAPI staining in blue. n = 3 independent experiments. Scale bars, 20 μm. (D) Quantification of the percentage of infected cells with nuclear TFEB using confocal microscopy from (C). n = 600 cells per condition from 3 independent experiments. (E) Quantification of the percentage of infected cells with nuclear TFE3 using confocal microscopy from (C). n = 600 cells per condition from 3 independent experiments. (F) Quantification of the p-S211-TFEB/total TFEB ratio represented as fold increase. n = 3 independent experiments. (G) Quantification of the p-S321-TFE3/total TFE3 ratio represented as fold increase. n = 3 independent experiments. (H) Calu-3 cells were infected with SARS-CoV-2 for the indicated times. Endogenous TFEB and TFE3 are shown in green and DAPI staining in blue. Scale bars, 20 μm. (I) Representative immunoblot analysis of protein lysates from HeLa-mCC1a cells infected with MHV for the indicated times. (J and K) Quantification of immunoblot data shown in (I). n = 3 independent experiments. (L) Representative western blot of TFEB and TFE3 levels in HeLa-mCC1a cells treated with either non-target or DCAF7 siRNAs for 72 h followed by infection with MHV for 16 h. (M) Quantification of immunoblot data shown in (L). n = 3 independent experiments. Statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparison post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data represent mean ± SEM.

Figure 2

Calcineurin regulates MHV-induced TFEB nuclear translocation (A) Representative images of the endogenous TFEB translocation assay obtained by confocal automated microscopy. HeLa-mCC1a cells were treated with MHV for 16 h. After 2 h of MHV infection, cells were treated with FK506 (PPP3/calcineurin inhibitor, 10 μM), BAPTA-AM (Ca²⁺ chelator, 10 μM), BIM IV (PKC inhibitor, 1 μm), CRT-0066101 (PKD inhibitor, 2.5 μM), and compound C (AMPK inhibitor, 2.5 μM). n = 3 independent experiments. Scale bars, 50 μm. (B) Quantification of average intensity of nuclear endogenous TFEB fluorescence. Automated imaging analysis included at least 10,000 cells per condition. n = 3 independent experiments. (C) Representative immunofluorescence images of endogenous TFEB (green) in HeLa-mCC1a cells treated with either non-target or PPP3/calcineurin catalytic subunits A and B siRNAs for 72 h followed by infection with MHV for 16 h. Cells were stained with DAPI (blue) n = 3 independent experiments. Scale bars, 20 μm. (D) Immunoblot analysis of protein lysates from HeLa-mCC1a treated with either non-target or PPP3/calcineurin catalytic subunits A and B siRNAs for 72 h followed by infection with MHV for 16 h n = 3 independent experiments. (E) Quantification of percentage of infected cells with nuclear TFEB using confocal microscopy from (C). n = 600 cells per condition from 3 independent experiments. (F) Representative immunofluorescence images of endogenous TFEB (green) in HeLa-mCC1a cells treated with either non-target or PPP2 catalytic subunits A and B siRNAs for 72 h followed by infection with MHV for 16 h. Cells were stained with DAPI (blue) n = 3 independent experiments. Scale bars, 20 μm (G) Quantification of percentage of infected cells with nuclear TFEB using confocal microscopy from (F). n = 300 cells per condition from 3 independent experiments. Statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparison post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data represent mean ± SEM.

Figure 3

TFEB and TFE3 depletion reduces the immune response induced by MHV infection (A) Principal-component analysis of genes with q-value <0.05 reveals distinct clustering of siControl- and siTFEB/TFE3-treated cells upon 24 h MHV infection. (B) Volcano plot indicating distribution of genes significantly up- (red) and downregulated (green) in control cells versus cells infected with MHV for 24 h. Cutoffs indicate genes with q-value <0.05. (C) Enriched gene ontology (GO) terms in the ‘MSigDB Hallmark’ category of differentially increased expression genes between control conditions and treated with MHV for 24 h. (D) Heatmap of differentially expressed immune response genes from RNA-Seq analysis of siControl versus siTFEB/3-treated cells in response to MHV infection. (E) Relative quantitative RT-PCR analysis of the mRNA expression of immune genes in HeLa-mCC1a cells treated with either control or TFEB/3 siRNAs upon incubation with MHV for 0 h, 6 h, 16 h, and 24 h n = 3 independent experiments. Statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparison post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data represent mean ± SEM.

Figure 4

TFEB and TFE3 induce expression of multiple immune genes (A) Schematic representations of the TFE3 binding region in the promoter of several immune genes in HeLa-mCC1a cells infected with MHV for 14 h. The transcription start site is indicated as TSS. (B) Relative quantitative RT-PCR analysis of the mRNA levels of the indicated immune genes in HeLa-mCC1a cells infected with either control adenovirus (Ad-null) or adenovirus expressing TFE3-321A (Ad-TFE3-S321A)for 40 h n = 3 independent experiments. (C) Relative quantitative RT-PCR analysis of the mRNA levels of the indicated immune genes in HeLa-mCC1a cells infected with either control adenovirus (Ad-null) or adenovirus expressing TFEB-S211A (Ad-TFEB-S211A) for 40 h n = 3 independent experiments. Statistical analysis with Student’s t-tests. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.

Figure 5

TFEB and TFE3 modulate cell viability in response to viral infection (A) Representative flow cytometry plots of HeLa-mCC1a cells treated with either siRNA Control (siControl) or siRNA against TFEB and TFE3 (siTFEB/TFE3) and infected with MHV for 16 and 24 h. Annexin V+ are apoptotic cells, 7-AAD + are necrotic cells, and Annexin V+/7-AAD + are late apoptotic and necrotic cells. (B) Quantification of the population of apoptotic cells (Annexin V+), necrotic cells (7-AAD+), and late apoptotic and necrotic cells (Annexin+/7-AAD+) from (A). n = 3 independent experiments. (C) Representative western blot of PARP1, Caspase-3, TFEB, and TFE3 from HeLa-mCC1a cells treated with siControl or siTFEB/TFE3 and then infected with MHV for 16 and 24 h n = 3 independent experiments. (D and E) Quantification of immunoblot data shown in (C). n = 3 independent experiments. (F) Quantification of LDH levels released to the extracellular media in HeLa-mCC1a cells treated with MHV for 16 h or 24 h. Cells were previously transfected with siRNA control (siControl) and siRNA against TFEB and TFE3 (siTFEB/3). In addition, cells were treated with a pan-caspase inhibitor Z-VAD after 2 h of MHV infection when indicated. n = 4 independent experiments. Statistical analyzes were performed with two-way ANOVA with Sıdak’s. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Values are mean ± SEM.

Figure 6

TFEB and TFE3 facilitate virus exit from cells and contribute to viral infectivity (A and B) HeLa-mCC1a cells were transfected with either non-target siRNAs (siControl) or siRNAs against TFEB and TFE3 (siTFEB/3) for 72 h and then infected with MHV for 16 h or 24 h. Representative immunofluorescence images showing surface LAMP1 levels. n = 3 independent experiments. Scale bars, 20 μm (B) Quantification of surface LAMP1 levels from (A). n = 30 cells per condition from 3 independent experiments. (C) Extracellular β-Hexosaminidase activity assay from siControl- or siTFEB/TFE3-treated HeLa-mCC1a cells following infection with MHV for 24 h n = 3 independent experiments. (D) Relative quantitative RT-PCR analysis of the MHV mRNA levels in the extracellular medium of HeLa-mCC1a transfected with non-target (siControl) or siRNA against TFEB and TFE3 (siTFEB/3) for 72 h and infected with MHV for 16 h or 24 h n = 3 independent experiments. (E) HeLa-mCC1a cells were transfected with non-target (siControl) or siRNA against TFEB and TFE3 (siTFEB/3) for 72 h and then treated with MHV for 16 h or 24 h. Then Supernatants were collected and reinoculated into new HeLa-mCC1a cells. After 48–72 h TCID50/mL was calculated. n = 3 independent experiments. (F) Model depicting the proposed role of TFEB and TFE3 in cellular response to beta-coronavirus. Statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparison post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data represent mean ± SEM.