Journal: bioRxiv

Article Title: Influenza A virus NS1 sequesters RNA:DNA hybrids to evade RNase H1-dependent innate immunity

doi: 10.64898/2026.05.04.722684

Figure Lengend Snippet: A . Schematic representation of the modified cytoDRIP-seq approach. A549 cells were infected with IAV at an MOI of 1 PFU/cell for 24h. Immunoprecipitations were performed with anti-V5 (control) or S9.6 antibodies. ΔDRIP-seq represents RNase H1-sensitive RNA:DNA hybrid signal calculated as non-RNase H1-treated samples minus RNase H1-treated samples. Three independent experiments were performed and analyzed. B-C. Total number of reads mapped to the (B) human genome and the (C) IAV genome. Data represent the composition of non-RNase H1-treated samples to illustrate the initial distribution of captured sequences. The dashed horizontal line indicates the minimum threshold for high-confidence signal detection (109 reads); data points below this line represent background noise or low-complexity libraries. Bars represent the mean of n=3 independent biological replicates, with individual dots indicating the values for each replicate. Error bars represent standard deviations. D-E . Bar plots showing the average distribution of normalized signal across human genomic features including coding sequence (CDS), exons, introns, untranslated regions (UTR), and intergenic regions. Data are represented as the mean fold change in signal density relative to the mock condition for (D) V5- and (E) S9.6-immunoprecipitated samples. Dots represent n=3 biological replicates derived from background-subtracted (ΔDRIP-seq) signal. Error bars represent standard deviations. F . Genomic distribution of viral-origin RNA:DNA hybrid sequences mapping to each of the eight IAV genome segments. Coverage plots show the ΔRPM signals (RPM difference between non-RNase H1-treated and RNase H1-treated samples; negative values set to zero) obtained from IAV-infected cells following immunoprecipitation with either S9.6 or V5 (control) antibodies. Overlaid traces in varying shades represent each of the n=3 independent replicates. Genome position is indicated on the x-axis.

Article Snippet: For immunoprecipitation, 10 μL Protein G Dynabeads (10004D; Thermo Fisher) were incubated with 10 μg of mouse monoclonal anti-RNA:DNA hybrid [S9.6] antibody (ENH001; Kerafast) or mouse anti-V5 antibody (MCA1360; Bio-Rad) in 1x binding buffer (20□mM Tris-HCl pH□8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate) for 4-6□h at 4°C.

Techniques: Modification, Infection, Control, Sequencing, Immunoprecipitation, Derivative Assay

Journal: bioRxiv

Article Title: Influenza A virus NS1 sequesters RNA:DNA hybrids to evade RNase H1-dependent innate immunity

doi: 10.64898/2026.05.04.722684

Figure Lengend Snippet: A-B . Immunofluorescence microscopy analysis (A) and associated colocalization analysis (B) of A549 cells infected, or mock, with IAV at an MOI of 1 PFU/cell for 24 h, followed by methanol fixation, proteinase K treatment, and staining for RNA:DNA hybrids (S9.6; green), NS1 (magenta), and DNA (DAPI; blue). Colocalization between NS1 and RNA:DNA hybrids was assessed by determining the Pearson’s correlation coefficient ( r ) between green and magenta signals. Boxplots indicate the minimum to maximum range (whiskers) and mean (horizontal line) r values from n=3 independent experiments. Each dot represents the r value from a single field of view. Non-merged images are presented in Supplementary Figure 3. C-D . Detection of dsRNA (9D5; green), NS1 (magenta), and DNA (DAPI; blue) (C), as well as corresponding colocalization analysis (D) in A549 cells treated as detailed in A-B. Non-merged images are presented in Supplementary Figure 3. E-F . Detection of RNA:DNA hybrids (S9.6; green), PB2 (magenta), and DNA (DAPI; blue) (E), as well as corresponding colocalization analysis (F) in A549 cells treated as detailed in A-B. Non-merged images are presented in Supplementary Figure 3. G-H . Detection of dsRNA (9D5; green), PB2 (magenta), and DNA (DAPI; blue) (G), as well as corresponding colocalization analysis (H) in A549 cells treated as detailed in A-B. Non-merged images are presented in Supplementary Figure 3. I . Immunoblot analysis following immunoprecipitation with specific antibodies. A549 cells were infected with IAV, or mock, at an MOI of 1 PFU/cell for 24 h followed by immunoprecipitation (IP) of resulting cell lysates with anti-V5, anti-dsRNA (9D5), or anti-RNA:DNA hybrid (S9.6) antibodies. Input cell lysate and IP fractions were analyzed by immunoblotting for NS1 and total IgG. J . Immunofluorescence microscopy analysis of A549 cells infected with wt IAV, IAV ΔNS1, or mock, at an MOI of 1 PFU/cell for 24 h, prior to processing as described in A. Upper row shows staining for RNA:DNA hybrids (S9.6; green), dsRNA (9D5; magenta), and DNA (DAPI; blue). Bottom row shows staining for IAV NP (red) and DNA (DAPI; blue). K . Quantification of cytoplasmic RNA:DNA hybrid signals (S9.6; green) from experiments described in J. Boxplots indicate the range and mean log 2 FC of the area fraction occupied by signal relative to mock-infected controls from n=3 independent experiments. Each dot represents the average log 2 FC from multiple fields imaged per well. Dotted line indicates no change relative to mock. For all panels, data are representative of at least n=3 independent experiments. For microscopy images, scale bars represent 10 µm. For panels B, D, F, and H, statistical significance was determined by Mann-Whitney U test (* P□ ≤□0.05; ** P□ ≤□0.01).

Article Snippet: For immunoprecipitation, 10 μL Protein G Dynabeads (10004D; Thermo Fisher) were incubated with 10 μg of mouse monoclonal anti-RNA:DNA hybrid [S9.6] antibody (ENH001; Kerafast) or mouse anti-V5 antibody (MCA1360; Bio-Rad) in 1x binding buffer (20□mM Tris-HCl pH□8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate) for 4-6□h at 4°C.

Techniques: Immunofluorescence, Microscopy, Infection, Staining, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: eLife

Article Title: Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity

doi: 10.7554/eLife.66501

Figure Lengend Snippet: ( A and B ) Protein complex of Tim-3 and NF90 overexpressed in cells. HEK293T cells were transfected with plasmids encoding HA-Tim-3, Flag-NF90, and Myc-Tim-3 for 24 hr, immunoprecipitated with HA or Flag antibody, respectively, and detected by western blot for the indicated antibodies. ( C and D ) Interaction of Tim-3 intracellular domain with NF90. Schematic structure of Tim-3 and the derivatives used are shown ( C ). Whole-cell lysis of HEK293T cells transfected with Flag-Tim-3 (WT), Flag-Tim-3 (Del), Flag-Tim-3 (4YF), and V5-NF90 was used for immunoprecipitation and immunoblotting, as indicated ( D ). ( E ) Immunoblot analysis of NF90. HEK293T cells were transfected with Tim-3 plasmid for 24 hr, and lysates were detected for NF90 expression by western blot (left). Peritoneal macrophages from wild-type (WT) and Tim-3-TG mice were lysed and NF90 protein was detected by western blot (right). The results shown in panels ( A, B, D, and E ) were performed three times. Figure 2—source code 1. Tim-3-WT were transfected into HEK293T cells. Figure 2—source code 2. Candidates interacts with Tim-3 . Figure 2—source data 1. Tim-3 interacts with NF90. Figure 2—source data 2. NF90 interacts with Tim-3 . Figure 2—source data 3. Tim-3 interacts with NF90 via intracellular tail. Figure 2—source data 4. Tim-3 inhibits NF90.

Article Snippet: Antibodies to β-actin, anti-Flag-Tag (CW0287), anti-HA-Tag (CW0092), anti-V5-Tag (CW0094), and anti-RFP (CW0253) were from purchased from CWBIO (China).

Techniques: Transfection, Immunoprecipitation, Western Blot, Lysis, Plasmid Preparation, Expressing

Journal: eLife

Article Title: Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity

doi: 10.7554/eLife.66501

Figure Lengend Snippet: ( A ) E3 ligases identified by mass spectrometry for top peptide hits (defined by Mascot score) associated with NF90 ubiquitination. ( B ) TRIM47 promotes NF90 degradation in a proteasome-dependent manner. Plasmids encoding Flag-NF90, HA-ubiquitin, along with increasing amounts of V5-TRIM47 (0.5, 1.0, and 2.0 ug), were transfected into HEK293T cells for 24 hr, cells were treated with and without MG132 (20 ug /ml), respectively, followed by western blot to examine the NF90 protein level. ( C ) Tim-3 accelerates TRIM47-mediated NF90 degradation in a dose-dependent manner. HEK293T cells were transfected with plasmids encoding Flag-NF90, HA-ubiquitin, and V5-TRIM47 and an increasing dose of plasmid encoding Red-Tim-3 (0.5, 1.0, and 2 ug) for 24 hr. The protein level of NF90 was examined in cells. ( D and E ) Tim-3 upregulates TRIM47 in protein levels. TRIM47 protein levels were analyzed by immunoblotting in lysates from wild-type (WT) or Tim-3KO and WT or Tim-3-TG macrophages infected with vesicular stomatitis virus (VSV) for the indicated time. ( F ) Tim-3 facilitates K63-linked ubiquitination mediated by TRIM47. Plasmids encoding HA-Ub-K63, V5-TRIM47, and Flag-Tim-3 were transfected into HEK293T cells. Cells were treated with MG132 (20 ug/ml) for 6 hr, and cell lysates were immunoprecipitated with Flag antibody and detected by western blot for K48-Ub levels. Three independent experiments were conducted for all panels. Figure 4—source code 1. Silence of TRIM47 in macrophages. Figure 4—source code 2. TRIM47 promotes NF90 degradation. Figure 4—source data 1. TRIM47 promotes NF90 degradation. Figure 4—source data 2. Tim-3 synthesizes with TRIM47 to promote NF90 degradation. Figure 4—source data 3. Tim-3 promotes TRIM47 expression. Figure 4—source data 4. Tim-3 promotes TRIM47 expression. Figure 4—source data 5. Tim-3promotes k63-linked ubiquitination of TRIM47.

Article Snippet: Antibodies to β-actin, anti-Flag-Tag (CW0287), anti-HA-Tag (CW0092), anti-V5-Tag (CW0094), and anti-RFP (CW0253) were from purchased from CWBIO (China).

Techniques: Mass Spectrometry, Transfection, Western Blot, Plasmid Preparation, Infection, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity

doi: 10.7554/eLife.66501

Figure Lengend Snippet: ( A and B ) The intracellular domain of Tim-3 and the DZF domain of NF90 interact with TRIM47 respectively. HEK293T cells were transfected with the indicated plasmids for 24 hr and treated with MG132 (20 ug/ml) for 6 hr. The cells were then lysed, immunoprecipitated with Tim-3 or Flag antibody, and detected by western blot using the indicated antibodies. ( C ) Tim-3 promotes NF90 degradation mediated by E3 ligase TRIM47. HEK293T cells were transfected with plasmids encoding HA-Ub-K48, V5-TRIM47, Flag-NF90, and Red-Tim-3, and treated with MG132 (20 ug/ml) for 6 hr. Cell lysates were then immunoprecipitated with Flag antibody and analyzed by western blot using the indicated antibodies. ( D ) Residue K297 of NF90 is the major site of TRIM47-mediated K48-linked ubiquitination. Flag-NF90-DZF (WT) or KR mutants, HA-Ub-K48 and V5-TRIM47, were transfected into HEK293T cells for 24 hr. Cells were then treated with MG132 (20 ug/ml) for 6 hr. Cell lysates were analyzed by western blot for K48-linked ubiquitination of NF90. ( E ) Residue K297 is the decisive site in TRIM47-mediated degradation of NF90. Plasmids encoding Flag-NF90-DZF (WT) or K297R mutants, PDsRed-Tim-3, HA-Ub-K48, and V5-TRIM47, were transfected into HEK293T cells, and cell lysates were examined by western blot for the indicated proteins. Three independent experiments were conducted for all panels. Figure 5—source code 1. Lys297 is a critical site for ubiquitination of NF9. Figure 5—source data 1. Tim-3 interacts with TRIM47 via its intracellular tail. Figure 5—source data 2. NF90 interacts with TRIM47 via its DZF domain. Figure 5—source data 3. Tim-3 promotes TRIM47 mediated ubiquitination of NF90. Figure 5—source data 4. Lys297 is a critical site for TRIM47-mediated ubiquitination of NF90. Figure 5—source data 5. Lys297 is a critical site for TRIM47-mediated ubiquitination of NF90-DZF.

Article Snippet: Antibodies to β-actin, anti-Flag-Tag (CW0287), anti-HA-Tag (CW0092), anti-V5-Tag (CW0094), and anti-RFP (CW0253) were from purchased from CWBIO (China).

Techniques: Transfection, Immunoprecipitation, Western Blot