Fig 1.
TRIM34 expression is upregulated by IFN treatment and IAV infection in human 293T and A549 cells.
Human 293T (A, C) and human A549 (B, D) cells were treated with recombinant universal type I IFN or infected with IAV during 24h. TRIM34 gene expression was evaluated by RT-qPCR (A, B) and by Western blot, using an anti-TRIM34 specific antibody, and an anti-GAPDH specific antibody, used as a loading control (C, D), and compared to the levels in mock-treated or mock-infected cells. Data in A and B represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA followed by Dunnett’s post-hoc test. Protein bands in C and D were quantified using the ImageJ software and normalized to the levels of GAPDH expression (numbers below the blots). TRIM34 positively affects IAV replication in vitro. (E) Human 293T cells were transfected with the pCAGGS plasmid expressing TRIM34-FLAG protein or a pCAGGS empty plasmid, as control. At 24h post-transfection (hpt), cells were mock-infected or infected with IAV (MOI 1). (F) Human 293T cells were transfected twice with a non-targeted (NT) control siRNA or TRIM34 siRNA at two consecutive days, 24 h apart. On day 3, cells were mock-infected or infected with IAV (MOI 1). (E, F) At 0-, 24-, and 48-hours post-infection (hpi), cell culture supernatants were collected and titrated by immunofocus assay in MDCK cells. Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05; Student’s t-test with Holm-Šídák correction.
Fig 2.
TRIM34 positively affects IAV replication in vitro.
(A) Schematic representation of recombinant PR8 viruses expressing Venus, mCherry or TRIM34. A modified IAV-PR8 NS segment encoding NS1, either Venus (top), mCherry (middle) or TRIM34-FLAG (bottom), and NEP are indicated. Orange boxes at the beginning and end of each viral segment represent the viral 3′ and 5′ noncoding regions (NCR). Blue and white boxes indicate the viral NS1 and NEP genes, respectively. The thosea asigna virus (TAV) 2A autoproteolytic cleavage site used for the expression of NS1 and Venus, mCherry or TRIM34 and the porcine teschovirus (PTV) 2A autoproteolytic cleavage site used for the expression of Venus, mCherry or TRIM34 and NEP are indicated in gray. (B, C, D, E) Human A549 or MDCK cells were mock-infected or infected (MOI 0,1 and 1) with the recombinant viruses (rIAV-Venus, rIAV-mCherry or rIAV-TRIM34). (B) At 24hpi, MDCK cells were fixed, permeabilized and visualized for Venus and mCherry expression. Cells were stained with anti-FLAG (TRIM34) and anti-NS1 (infected-cells control) antibodies. DAPI was used for nuclear staining. (C-D) MDCK cells were infected with the viruses rIAV-Venus, rIAV-mCherry and rIAV-TRIM34 at MOIs 0.1 (C) or 1 (D) and viral titers were determined at 48 and 72hpi in MDCK cells. (E) A549 cells were infected with the viruses rIAV-Venus, rIAV-mCherry and rIAV-TRIM34 at MOI 1, and viral titers were determined at 48hpi in MDCK cells. Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. **p < 0.01, ***p < 0.001, ****p < 0.0001 for comparisons between rIAV-TRIM34 with rIAV-Venus and rIAV-mCherry using one-way ANOVA followed by Dunnett’s post-hoc test.
Fig 3.
TRIM34 protein interacts with the nucleocytoplasmic transport machinery.
(A) Human 293T cells were co-transfected with the pCAGGS plasmids encoding TRIM34-FLAG or the empty plasmid, as control. At 24hpt, cells were mock-infected or infected with IAV (MOI 1). Co-immunoprecipitation (co-IP) experiments using agarose beads conjugated to an anti-FLAG antibody, to pull down TRIM34 were performed. TRIM34 was detected by Western blotting using a TRIM34-specific antibody in the cellular lysates (input) and after the co-IP. An anti-GAPDH antibody served as a loading control for the input samples. Molecular weight markers (in kilodaltons) are indicated on the right. (B) Venn diagram of proteins identified by mass spectrometry in the co-IP pull-downs. (C) Qualitative proteomic analysis of specific proteins following TRIM34 overexpression. Gene Ontology (GO) biological processes were identified using the DAVID functional annotation tool and selected based on statistical significance (FDR < 0.05). The graphical representation includes GO terms containing the keywords ‘nucleus’, ‘nuclear’, or ‘nucleocytoplasmic’, with fold enrichment (>2) and gene counts (>3), shown for both mock and IAV conditions. The X-axis represents fold enrichment, with the number of genes indicated next to each bar.
Table 1.
Common host proteins associated with nuclear transport processes found in the TRIM34-interactome, both in mock and IAV conditions. Proteins identified in both mock and IAV-infected samples that belong to the selected GO biological processes. The common processes include nucleocytoplasmic transport, mRNA export from the nucleus, and protein import into the nucleus.
Fig 4.
TRIM34 interacts with Nup93 and reduces IRF3-5D nuclear translocation.
(A, B) Human 293T cells were co-transfected with the plasmid pEGFP-C1X expressing Nup93 fused to the EGFP protein alone or together with the pCAGGS plasmid encoding TRIM34-FLAG. At 24hpt, cells were (A) mock-infected o (B) infected with IAV (MOI 1). (A, B) Co-immunoprecipitation (Co-IP) experiment using an anti-FLAG antibody to pull down TRIM34 were performed. TRIM34 and Nup93 were detected by Western blotting using specific antibodies for the FLAG tag (to detect TRIM34) and the GFP tag (to detect Nup93) in the cellular lysates (input) and after the co-IP. An anti-GAPDH antibody served as a loading control for the input samples, and an anti-IAV NP antibody was used to confirm infection. Molecular weight markers (in kilodaltons) are indicated on the right. (C) Human 293T cells were co-transfected with the TRIM34-FLAG-encoding plasmid alone or together with the Nup93-myc-encoding plasmid. A co-immunoprecipitation (Co-IP) experiment using an anti-myc antibody to pull down Nup93 was performed. TRIM34 and Nup93 were detected by Western blotting using specific antibodies for the FLAG tag (to detect TRIM34) and the myc tag (to detect Nup93) in the cellular lysates (input) and after the co-IP. An anti-GAPDH antibody served as a loading control for the input samples. Molecular weight markers (in kilodaltons) are indicated on the right. (D) Human 293T cells were co-transfected with the plasmid pEGFP-C1X expressing Nup93 fused to the EGFP protein alone or together with the pCAGGS plasmid encoding TRIM34-FLAG. At 24hpt, cells were fixed with paraformaldehyde, TRIM34 was detected using an anti-FLAG antibody (red), Nup93 was identified through the GFP fluorescence signal encoded by the plasmid (green), and nuclei were stained with DAPI (blue). Confocal images show cells transfected with the plasmid expressing TRIM34-FLAG and Nup93-GFP separately, and cells co-transfected with both plasmids. Areas of co-localization of both proteins appear in yellow in the third picture and in white in the fourth picture (zoom). Scale bar, 10 μm. (E) Pearson’s correlation coefficient for Nup93 and TRIM34 fluorescent signals. Data represents means and SDs of 30 cells that were counted in three independent experiments. (F) Human HeLa cells were co-transfected with the pCAGGS plasmids encoding TRIM34-FLAG or the empty control, and the pCAGGS-IRF3-5D-GFP, expressing a constitutively active form of IRF3 (IRF3-5D) fused to GFP [63,64]. At 24hpt, cells were fixed with paraformaldehyde, TRIM34-FLAG was labeled with a specific antibody for the tag (in red) and nuclei were stained with DAPI (in blue). Scale bar, 10 μm. (G) Fluorescence intensity of IRF3-5D-GFP was quantified in the nucleus and cytoplasm to determine nuclear-to-cytoplasmic (N/C) ratios. (F, G) Data represents means and SDs of 58 (empty) and 76 (TRIM34) cells that were counted in three independent experiments. ****p < 0.0001; Student’s t-test with Holm-Šídák correction.
Fig 5.
Human 293T cells were co-transfected with the pcDNA3.1-RAE1-myc plasmid, expressing RAE1 fused to a c-myc tag, alone or in combination with the pCAGGS plasmid encoding TRIM34-FLAG or with the pCAGGS plasmid encoding GBP1-FLAG. At 24hpt, (A) cells were mock-infected or (B) infected with IAV (MOI 1). In addition, cells were co-transfected with the plasmids pcDNA3.1-RAE1-myc and pcDNA3.1-GBP1-FLAG to confirm that the interaction between TRIM34 and RAE1 is not due to the FLAG tag (B). (A, B) Co-immunoprecipitation (Co-IP) experiments using an anti-FLAG antibody, to pull-down TRIM34 were performed. TRIM34, GBP1, and RAE1 were detected by Western blotting using specific antibodies for the FLAG tag (to detect TRIM34 and GBP1) and the c-myc tag (to detect RAE1) in the cellular lysates (input) and after the IP. An anti-GAPDH antibody served as a loading control for the input samples, and an anti-IAV NP antibody was used to confirm infection. Molecular weight markers (in kilodaltons) are indicated on the right. (C, D) Co-immunoprecipitation (Co-IP) experiments using an anti-c-myc antibody, to pull-down RAE1 were performed. 293T cells were transfected with TRIM34-FLAG-overexpressing plasmid together with the RAE-1-myc-overexpressing plasmid (C), or with the RAE1-myc overexpressing plasmid alone (D). TRIM34, GBP1, and RAE1 were detected by Western blotting using FLAG antibody (to detect TRIM34 and GBP1) and c-myc antibody (to detect RAE1) in input lysates and after IP (C), or with anti-TRIM34 specific antibody replacing anti-FLAG antibody (D). GAPDH was used as a loading control for the input samples, and an anti-IAV NP antibody was used to confirm infection. Molecular weight markers (in kilodaltons) are indicated on the right. Partial co-localization of TRIM34 and RAE1 in the cytoplasm. (E) At 24hpt, cells were fixed with paraformaldehyde, TRIM34-FLAG and RAE1-myc were labeled with specific antibodies for the tags (in red and yellow, respectively), and nuclei were stained with DAPI (in blue). Confocal images show cells transfected with the plasmid expressing TRIM34-FLAG and RAE1-myc separately, and cells co-transfected with both plasmids. Areas of co-localization of both proteins appear in orange in the third picture and in white in the fourth picture (zoom). Scale bar, 10 μm. (F) Pearson’s correlation coefficient for RAE1 and TRIM34 fluorescent signals. Data represents means and SDs of 30 cells that were counted in three independent experiments. TRIM34 decreases host gene expression. (G) Human 293T cells were co-transfected with a plasmid constitutively expressing Renilla luciferase (Rluc) and plasmids encoding TRIM34-FLAG, GBP1-FLAG or RAE1-myc alone, or the plasmids expressing TRIM34-FLAG and RAE1-myc together. At 24hpt, cells were mock-infected and infected with IAV (MOI 1). (G) At 24hpi, levels of Rluc were determined and normalized to the levels of mock-infected cells transfected with the empty plasmid or to the levels of IAV-infected cells transfected with the empty plasmid. (H) Alternatively, cells were co-transfected with a plasmid expressing Gaussia luciferase (Gluc) and the plasmid encoding TRIM34-FLAG or an empty plasmid, as control, and the cells were left mock-infected or infected with IAV. At 24 hpi, secreted and intracellular levels of Gluc were quantified and normalized to the levels in cells transfected with the empty plasmid. (G, H) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. (G) **p < 0.01, ***p < 0.001; one-way ANOVA followed by Dunnett’s post-hoc test. (H) *p < 0.05; Student’s t-test with Holm-Šídák correction.
Fig 6.
TRIM34 impairs innate immune responses induced by IFN.
(A, B) Human 293T cells were transfected with the plasmid pCAGGs-TRIM34-FLAG or the empty control plasmid. (A) At 24h post-transfection, cells were non-treated or treated with type I IFN. At 24h post-IFN-treatment, CXCL10, IFNB1, IFNL1 and IFIT2 expression was measured by RT-qPCR and mRNA levels were expressed as fold-change (increases) in comparison to mock-treated cells transfected with the empty plasmid, used as control. (B) At 24h post-IFN-treatment, cells were infected with rVSV-GFP (MOI 0.1) and viral titers were measured by lysis plaque assay at 24hpi. Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05 for comparisons between empty and TRIM34 overexpressed cells using Student’s t-test with Holm-Šídák correction. Human 293T cells (C, F), human lung adenocarcinoma-derived cells A549 cells (D, G), or the non-tumorigenic human bronchial epithelial BEAS-2B (E, H) were transfected twice with NT control siRNA or TRIM34 siRNA, 24h apart, for two consecutive days. On day 3, cells were treated with IFN for an additional 24h. (C, D, E) TRIM34, and (F, G, H) CXCL10, IFNB1, IFNL1 and IFIT2 expression was measured by RT-qPCR and mRNA levels were expressed as fold-change (increases) in comparison to mock-treated cells, transfected with the NT siRNA, used as control. (A-H) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05 for comparisons between control and TRIM34 knocked-down cells using Student’s t-test with Holm-Šídák correction.
Fig 7.
TRIM34 impairs innate immune responses induced by IAV.
(A) Human 293T cells were transfected with the pCAGGS plasmids encoding TRIM34-FLAG or the empty plasmid, as control. At 24h post-transfection, cells were mock-infected or infected with IAV or IAV-ΔNS1. At 24hpi, the levels of CXCL10, IFNB1, IFNL1, and IFIT2 mRNAs were evaluated by RT-qPCR, and expressed as fold-change (increases) in comparison to mock-infected cells, transfected with the empty plasmid, used as control. (B) Human 293T cells were transfected twice with the NT control siRNA or TRIM34 siRNA every 24h for two consecutive days. On day 3, cells were infected with IAV or IAV-ΔNS1 (MOI 1) for an additional 24h. The levels of CXCL10, IFNB1, IFNL1 and IFIT2 were measured by RT-qPCR and mRNA levels were expressed as fold- change (increases) in comparison to mock-infected cells transfected with the NT siRNA, used as controls. Human A549 (C) or BEAS-2B (D) cells were transfected twice with the NT control siRNA or TRIM34 siRNA every 24h for two consecutive days. On day 3, cells were infected with IAV for an additional 24h. (B, C, D) The levels of CXCL10, IFNB1, IFNL1 and IFIT2 were measured by RT-qPCR and mRNA levels were expressed as fold change (increases) in comparison to mock-infected cells transfected with the NT siRNA, used as controls. (A-D) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05 for comparisons between empty and TRIM34 overexpressed cells, or NT siRNA and TRIM34 knocked-down cells, using Student’s t-test with Holm-Šídák correction. (E, F) A549 cells were infected with the viruses rIAV-Venus, rIAV-mCherry and rIAV-TRIM34 at MOI 1, and the levels of (E) IAV NP-vRNA, and (F) CXCL10, CCL2, IFNL1 and IFIT2 mRNAs were determined at 24hpi. (E, F) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05, **p < 0.01, ***p < 0.001 for comparisons between rIAV-TRIM34 with rIAV-Venus and rIAV-mCherry using one-way ANOVA followed by Dunnett’s post-hoc test.
Fig 8.
Effect of TRIM34 expression on the induction of innate immune responses in vivo.
(A, B, and C) Mice (n = 5/group) were infected with the recombinant viruses rIAV-mCherry and rIAV-TRIM34 (2,000 FFU/mice). (A) Weight loss was monitored daily for 14 days post-infection. Differences in body weights between rIAV-mCherry and rIAV-TRIM34-infected mice were analyzed by two-way ANOVA; ***p < 0.001, ***p < 0.0001. (B) At 24, and 48 hpi, viral titers in mouse lungs were determined, and (C) CCL2, CXCL10, IFNB1, IFNL3, IL-1b and IFIT2 mRNA expression was evaluated in mouse lungs by RT-qPCR. Each dot corresponds to one rIAV-infected mouse. Increases in mRNA levels were expressed as fold-changes in comparison to mock-infected mice, used as controls. *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA followed by Tukey’s post-hoc test.
Fig 9.
TRIM34 limits nuclear export of immune-regulated mRNAs.
Human 293T cells were transfected with the pCAGGS plasmids encoding TRIM34-FLAG or the empty plasmid, as control. At 24h post-transfection, cells were treated with IFN or infected with IAV. At 24hpi, cells were fractionated into cytoplasm and nuclear RNA fractions. (A) Nuclear/Cytoplasmic (N/C) ratios of CXCL10, IFNB1, IFNL1, IFIT2, IFI27 and (B) IAV PB2, HA, NP and NA-mRNAs were quantified in TRIM34-overexpressed cells and in empty-plasmid transfected cells. (A, B) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05 or ns (not-statistically significant) for comparisons between empty and TRIM34 overexpressed cells using Student’s t-test with Holm-Šídák correction.
Fig 10.
E3 ubiquitin ligase activity is not essential for TRIM34 effect on suppressing host gene expression or its interaction with RAE1.
(A) Comparison of the first 60 amino acids in the RING domain between rhesus macaque TRIM5α and human TRIM34 proteins, highlighting sequence homology (in bold). The three selected mutants were: I17A/L19A (red), E20K (purple) and H32A (green). Self-ubiquitylation of TRIM34. (B) Human 293T cells were co-transfected with a plasmid expressing ubiquitin-HA together with the pCAGGS plasmids encoding the FLAG-tagged TRIM34 variants (H32A, E20K and I17L/L19A), the wild-type TRIM34 protein, or the empty plasmid, as control. An immunoprecipitation experiment using an anti-FLAG antibody, to pull down TRIM34 was performed. Ubiquitylated TRIM34 was analyzed by Western blotting with the anti-HA antibody and the expression of WT, H32A, E20K and I17L/L19A TRIM34 variants was detected using the anti-FLAG antibody in the cellular lysates (input) and after the co-IP. Molecular weight markers (in kilodaltons) are indicated on the right. Ubiquitylated TRIM34 protein bands were quantified using the ImageJ software and normalized to the levels of anti-FLAG (numbers below the blots). (C) Human 293T cells were co-transfected with a plasmid constitutively expressing Rluc and plasmids encoding the empty control, TRIM34 WT or TRIM34 mutants (H32A, E20K, I17L/L19A). At 24hpt, levels of Rluc were determined and normalized to the levels of cells transfected with the empty plasmid. Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. ****p < 0.0001 for comparisons between empty and TRIM34 (WT or mutants) overexpressed cells using one-way ANOVA followed by Dunnett’s post-hoc test. (D) Human 293T cells were co-transfected with the pCAGGS plasmids encoding FLAG-tagged mutants (H32A, E20K and I17L/L19A) or wild-type TRIM34 (WT), and the pcDNA3.1-RAE1-myc, expressing RAE1 fused to a c-myc tag. Co-IP experiments using an anti-FLAG antibody, to pull-down TRIM34 were performed. The expression of TRIM34 (WT or mutants) was analyzed by Western blotting using the anti-FLAG antibody and RAE1 was detected with the c-myc tag antibody in the cellular lysates (input) and after the co-IP. Molecular weight markers (in kilodaltons) are indicated on the right.