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: 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.

Fig 4: 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 5: TRIM34 interacts with RAE1.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.