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