Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Luciferase activity in A549 cells transfected with siRNAs targeting paraspeckle components. Data are represented as mean ± SD (n = 3). siNEAT1_1 and 2 indicate two different siRNAs both targeting the whole NEAT1.The data for RBM14, RBMX, RBM5, HNRNPA1, HNRNPA2B1, HNRNPK and PSPC1 were reproduced from Figure S4C. (B) Quantitative PCR analysis of viral RNA species of fragment NP normalised to GAPDH in A549 cells with knockdowns of paraspeckle proteins, infected with WSN (MOI 3) at 6hpi. Data are presented as log-fold-change of as mean ± SD relative to the siNT condition (n=3). (C) Schematic of NONO knockout and rescue workflow. CRISPR-Cas9 with two sgRNAs generated NONO KO1 and KO2 A549 cell lines. Lentiviral overexpression of mEGFP-NONO or mEGFP (control) in KO cells was used for rescue. Wild-type, NONO KO, and rescued cells were analysed for paraspeckle function and viral replication. Created in BioRender. https://BioRender.com/t41h404 . (D) Luciferase activity in wt, NONO KO1, and KO2 in A549 cells (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (E) Luciferase activity in wt, NONO KO1, and KO2 A549 cells lentivirally overexpressing mEGFP (control) or mEGFP-NONO (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (F) Density plot of log2-fold-changes of proteins cross-linked to NONO, NP and NS1 identified by AP-MS (NONO infected vs. NONO mock). Proteins cross-linked to NP and NS1 (orange) are co-depleted in infected cells compared to proteins not linked to NP or NS1 (grey). (G) Log2-fold changes of interactors identified by AP-MS against NONO (infected vs. mock) and NP (infected vs. infected isotype control). Proteins cross-linked to NONO (pink), NP (orange), and non-associated proteins (grey) are shown. (H) Selected protein categories enriched in both NP and NONO-mock AP-MS datasets from G (see also Table S3). (I) Schematic representation of paraspeckles disruption: IAV proteins, particularly NP and NS1, disrupt paraspeckle integrity by binding core proteins like SFPQ and NONO, and possibly NEAT1, initiating paraspeckle disassembly. As the infection progresses, PA-X promotes NEAT1 degradation, while POL II inhibition further destabilises paraspeckles, leading to their complete disruption. Created in BioRender. https://BioRender.com/y54j344 . (A-B, D-E) Data are represented as mean ± SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Infection, Knock-Out, CRISPR, Generated, Over Expression, Control, Disruption, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Cross-linking network between paraspeckle and viral proteins. Pink – essential for paraspeckle formation, blue – important, yellow – localised to paraspeckles but dispensable , gray – other proteins cross-linked to both paraspeckle and orange - viral proteins. (B) Paraspeckle structure, showing the interaction between NEAT1 long noncoding RNA and proteins such as SFPQ and NONO (left) and the NEAT1 isoforms, NEAT1_1 and NEAT1_2, and the position of FISH probes/qPCR primers on NEAT1 used in this study (right). Created in bioRender. https://BioRender.com/b92y974 (C) AP-MS of NONO versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). NONO and its known interactors are coloured pink and viral proteins - orange. (D) AP-MS of NP versus IgG controls from WSN-infected A549 cells (14 hpi, n=3). Proteins that were also identified in SHVIP are highlighted. Proteins coloured as in E. (E) Maximum projection of confocal microscopy images of A549 cells infected with WSN (MOI 3) at 4, 8, and 12 hpi. NEAT1 - magenta, vRNA (PB2 fragment) - green, and DNA (DAPI) - grey. (F) CV of NEAT1_2 (NEAT1_1 in case of MEF) in the nucleus across different cell lines infected with WSN. (G) Number of paraspeckles per nucleus across different cell lines infected with WSN. (F-G) Data are represented as mean + SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Infection, Confocal Microscopy

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Luciferase activity in A549 cells transfected with siRNAs targeting paraspeckle components. Data are represented as mean ± SD (n = 3). siNEAT1_1 and 2 indicate two different siRNAs both targeting the whole NEAT1.The data for RBM14, RBMX, RBM5, HNRNPA1, HNRNPA2B1, HNRNPK and PSPC1 were reproduced from Figure S4C. (B) Quantitative PCR analysis of viral RNA species of fragment NP normalised to GAPDH in A549 cells with knockdowns of paraspeckle proteins, infected with WSN (MOI 3) at 6hpi. Data are presented as log-fold-change of as mean ± SD relative to the siNT condition (n=3). (C) Schematic of NONO knockout and rescue workflow. CRISPR-Cas9 with two sgRNAs generated NONO KO1 and KO2 A549 cell lines. Lentiviral overexpression of mEGFP-NONO or mEGFP (control) in KO cells was used for rescue. Wild-type, NONO KO, and rescued cells were analysed for paraspeckle function and viral replication. Created in BioRender. https://BioRender.com/t41h404 . (D) Luciferase activity in wt, NONO KO1, and KO2 in A549 cells (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (E) Luciferase activity in wt, NONO KO1, and KO2 A549 cells lentivirally overexpressing mEGFP (control) or mEGFP-NONO (WSN with PB2-T2A-NanoLuc, MOI 0.01, 48 hpi). Data are represented as mean ± SD (n = 3). (F) Density plot of log2-fold-changes of proteins cross-linked to NONO, NP and NS1 identified by AP-MS (NONO infected vs. NONO mock). Proteins cross-linked to NP and NS1 (orange) are co-depleted in infected cells compared to proteins not linked to NP or NS1 (grey). (G) Log2-fold changes of interactors identified by AP-MS against NONO (infected vs. mock) and NP (infected vs. infected isotype control). Proteins cross-linked to NONO (pink), NP (orange), and non-associated proteins (grey) are shown. (H) Selected protein categories enriched in both NP and NONO-mock AP-MS datasets from G (see also Table S3). (I) Schematic representation of paraspeckles disruption: IAV proteins, particularly NP and NS1, disrupt paraspeckle integrity by binding core proteins like SFPQ and NONO, and possibly NEAT1, initiating paraspeckle disassembly. As the infection progresses, PA-X promotes NEAT1 degradation, while POL II inhibition further destabilises paraspeckles, leading to their complete disruption. Created in BioRender. https://BioRender.com/y54j344 . (A-B, D-E) Data are represented as mean ± SD. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Infection, Knock-Out, CRISPR, Generated, Over Expression, Control, Disruption, Binding Assay, Inhibition