human a549 lung epithelial cells  (ATCC)

Journal: bioRxiv

Article Title: Influenza virus antagonizes self sensing by RIG-I to enhance viral replication

doi: 10.1101/2025.03.12.642847

Figure Lengend Snippet: (A) WT A549 cells or those stably expressing RIG-I were mock-infected, infected with FLUAV (MOI = 0.01, 24h), or infected with VSV-GFP (MOI = 0.001, 24h). Protein expression was detected by western blotting. (B) RIG-I CLIP was performed with a spike-in control in biological duplicate. Fraction of reads mapping to human, FLUAV, and VSV genomes for size-matched input controls or RIG-I CLIP samples. (C) Normalized read coverage for RIG-I-bound and size-matched input control RNAs. Data from independent biological replicates are shown as mean and s.d. using dark and light shades of the same color, respectively. CLIPper-identified peaks are shown. CPM, counts per million. See Supplementary Table 1 for all identified RIG-I CLIP peaks. (D) Gene enrichment in RIG-I binding during infection. Log2 fold-change in read depth of genes captured by RIG-I CLIP versus the size- matched input control plotted as a function of the average counts per million (CPM) in the CLIP and size-matched input libraries. Differential gene binding data can be found in Supplementary Table 2. See also Supplementary Table 3 for differential peak binding. (E) Principle coordinate analysis of genes identified in the spike-in-normalized RIG-I CLIP and size-matched input libraries. (F) Overlap of CLIPper-called RIG-I binding peaks from mock, FLUAV-infected, and VSV- infected A549 cells. (G) Normalized read coverage of RIG-bound RNAs or size-matched input controls from mock, FLUAV-infected, and VSV-infected cells shown as in (C). CLIPper-identified peaks and gene tracks are shown. (H) Log2 fold-change of infection over mock for RIG-I CLIP peak intensity separated by host- or virus-derived RNAs. Associated with Supplementary Table 3.

Article Snippet: Human lung alveolar epithelial (A549) cells (CCL-185), Madin-Darby canine kidney (MDCK) cells (CCL-34), baby hamster kidney-21 (BHK-21) cells (CCL-10), and human embryonic 293T (HEK293T) cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

Techniques: Stable Transfection, Expressing, Infection, Western Blot, Control, Binding Assay, Virus, Derivative Assay

Journal: bioRxiv

Article Title: Influenza virus antagonizes self sensing by RIG-I to enhance viral replication

doi: 10.1101/2025.03.12.642847

Figure Lengend Snippet: (A-B) NP interacts with RIG-I in an RNA-dependent manner. Cells expressing Strep-tagged RIG-I or an mCherry control were infected with 2009 pH1N1 influenza virus, lysed and subject to enrichment with Strep-tag affinity resin. Proteins in the input lysate and enriched samples were detected by western blot. In (B), samples were subject to RNAse A treatment prior to affinity capture. (C) RNA-immunoprecipitation was performed on lysates prepared from A549 cells infected for 24h with A/WSN/33 (MOI = 0.02) using antibody recognizing NP or an IgG control. RNAs were subject to Bioanalyzer analysis and NP was detected by western blot. (D) Frequency of host- and FLUAV-derived reads in SMInput and NP eCLIP libraries prepared from A549 cells infected with FLUAV for 24h (MOI = 0.02). (E) Identification of NP-bound gene transcripts during infection. Log2 fold-change in read depth of genes captured by NP CLIP versus the size-matched input control plotted as a function of the average counts per million (CPM) in the CLIP and size-matched input libraries. Negative-sense vRNA and positive- sense cRNA viral gene segments and select host genes are indicated. Differential gene binding data can be found in Supplementary Table 4. (F) Normalized read coverage for NP-bound and size-matched input control RNAs. Data from independent biological replicates are shown as mean and s.d. using dark and light shades of the same color, respectively. CLIPper-identified peaks and gene tracks are shown. CPM, counts per million. See Supplementary Table 5 for all identified NP CLIP peaks. (G) Human RNA biotypes of CLIPper-called peaks (left) and further division of non-coding RNAs (ncRNA) into annotated biotypes (right). (H) Read counts in CLIPper-called peak were assigned to host-derived protein-coding or non-coding/repetitive RNAs based on their RNA biotype. (I) Clustering of eCLIP datasets from ENCORE database with uniform manifold approximation and projection based on Jaccard similarity. Dot color denoted by NP eCLIP library (red) or available hierarchical clustering of eCLIP libraries in the ENCORE database.

Article Snippet: Human lung alveolar epithelial (A549) cells (CCL-185), Madin-Darby canine kidney (MDCK) cells (CCL-34), baby hamster kidney-21 (BHK-21) cells (CCL-10), and human embryonic 293T (HEK293T) cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

Techniques: Expressing, Control, Infection, Virus, Strep-tag, Western Blot, Capture-C, RNA Immunoprecipitation, Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: Influenza virus antagonizes self sensing by RIG-I to enhance viral replication

doi: 10.1101/2025.03.12.642847

Figure Lengend Snippet: (A) Expression of NP in tetNP-A549 cells via doxycycline induction, infection with FLUAV (MOI = 0.01. 24 h), or both. Proteins were detected by western blotting. PB2 served as a control for successful infection control. (B) NP CLIP was performed for the indicated conditions using a shared spike-in control. Frequency of human- and FLUAV-derived reads was determined for NP CLIP and size- matched input control libraries. (C) Principal coordinate analysis of spike-in- normalized NP CLIP and size-matched input control libraries. (D) Overlap of NP-binding peaks for each condition. (E) Normalized read coverage for NP-bound and size-matched input control RNAs. Data from independent biological replicates are shown as mean and s.d. using dark and light shades of the same color, respectively. CLIPper-identified peaks and gene tracks are shown. CPM, counts per million. Associated with Supplemental Tables 6-7. (F). Log2 CPM of select non-coding RNAs in the size-matched input control. (G) Immunofluorescence microscopy of tetNP-A549s stained for NP 24 h after infection (MOI = 0.01) or doxycycline induction. (H) Read coverage as in (E) over the MALAT1 gene body.

Article Snippet: Human lung alveolar epithelial (A549) cells (CCL-185), Madin-Darby canine kidney (MDCK) cells (CCL-34), baby hamster kidney-21 (BHK-21) cells (CCL-10), and human embryonic 293T (HEK293T) cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

Techniques: Expressing, Infection, Western Blot, Control, Derivative Assay, Binding Assay, Immunofluorescence, Microscopy, Staining

Journal: bioRxiv

Article Title: Influenza virus antagonizes self sensing by RIG-I to enhance viral replication

doi: 10.1101/2025.03.12.642847

Figure Lengend Snippet: (A) Overlap of the union of RIG-I CLIP peaks identified in all conditions in in with NP CLIP peaks identified in infected cells in . (B) Read coverage on 7SL RNA of the 5’-mapped base in RIG-I (top) and NP (bottom) CLIP libraries. Coverage was normalized in each library to enable direct comparisons across samples. Data from independent biological replicates are shown as mean and s.d. using dark and light shades of the same color, respectively. CLIPper-identified peaks and gene tracks are shown. (C) Minimum free energy RNA secondary structures predictions from RNAFold for select non-coding RNAs. (D) RNA was isolated from RNA immunoprecipitations (RIPs) performed with the indicated antibodies in cell lysates where NP was expressed by infection (infect) or transfection (tx). ISRE reporter cells were transfected with the recovered RNAs and ISRE activation was measured relative to mock-treated cells. Total eukaryotic RNA or IFN-β were included as controls. Data normalized to an internal Renilla control and plotted relative to mock-treated cells. Red arrowhead = lack of activation by RNAs bound by NP in transfected cells. (E) Eluates of NP RNA-immunoprecipitations from FLUAV infected A549 cells (MOI = 0.02, 24h) were treated with an RNaseH-based depletion targeting influenza RNA or mock treated. Remaining RNAs were transfected into WT or RIG knockout ( DDX58 -/- ) A549 ISRE-reporter cells. ISRE-promoter induction was normalized to an internal Renilla control and shown relative to mock-treated conditions for each cell line. (F) WT, RIG knockout ( DDX58 -/- ) , or MAVS knockout ( MAVS -/- ) A549 ISRE-reporter cells were transfected with decreasing amounts of in vitro transcribed non-coding RNAs, mixed molecular weight poly IC, mock transfected, or treated with IFN-β. ISRE-promoter induction shown relative to mock-treated conditions for each cell line. (G) Poly IC or in vitro transcribed non-coding RNAs were treated with calf-intestinal phosphatase (CIP) or mock treated and transfected into ISRE-reporter cells. Cells were assayed for ISRE-induction and normalized to mock-transfected cells. * P < 0.05, *** P < 0.001, **** P < 0.0001 with one-way ANOVA and Dunnet’s post-hoc correction for (D) and a two-way ANOVA with Tukey’s multiple comparison test in (E-G).

Article Snippet: Human lung alveolar epithelial (A549) cells (CCL-185), Madin-Darby canine kidney (MDCK) cells (CCL-34), baby hamster kidney-21 (BHK-21) cells (CCL-10), and human embryonic 293T (HEK293T) cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

Techniques: Infection, Isolation, Transfection, Activation Assay, Control, Knock-Out, In Vitro, Molecular Weight, Comparison

Journal: bioRxiv

Article Title: Influenza virus antagonizes self sensing by RIG-I to enhance viral replication

doi: 10.1101/2025.03.12.642847

Figure Lengend Snippet: (A) Antisense oligos (ASO) were used to purify the indicated RNAs from infected or mock treated cells. Heatmap of qRT-PCR results showing highly specific enrichment of desired RNAs. Enrichment is shown relative to RNA levels in the input from infected cells. (B) A549 ISRE- or IFN-β-reporter cell lines were transfected with ASO-purified RNAs from mock-treated or infected cells described in (A). Induction is relative to mock-transfected cells. There were no significant differences when RNAs were purified from uninfected cells. (C) 5’ PPP RNAs were marked by vaccinia capping enzyme, purified, and quantified by RT-qPCR. RNAs from influenza virus-infected A549 cells were compared to mock-treated cells. (D) NP or the NLS-mutant NP K7A/R8A was expressed in 293T ISRE-firefly reporter cells for 24 h prior to transfection with Y4 RNA or mock-transfection. ISRE induction was measured relative to an internal Renilla luciferase control. (E) WT or RIG-I knockout ( DDX58 -/- ) A549 cells were transfected with the decreasing concentrations of non-coding RNA, poly IC, or mock transfected. Cells were subsequently infected with an A/WSN/33-based reporter virus. Supernatants were recovered 24 hpi and titered on MDCK cells. (F) A model for viral antagonism of self sensing. Under basal conditions, RIG-I rapidly samples and recycles off of non-immunogenic host RNAs maintaining a pool of RIG-I that surveils the entire RNA landscape. During infection, immunogenic features on host RNAs promote RIG-I oligomerization and activation of innate immune pathways that suppress viral infection. Influenza virus NP counters this by binding host RNAs, interfering with RIG-I activation or recycling to promote viral replication. * P<0.05, ** P < 0.01 and **** P < 0.0001 as determined by one-way ANOVA (C) or two-way ANOVA (B, D, E) with post hoc Šídák’s multiple comparisons test. Comparison were made to mock cells in each condition. ns = not significant.

Article Snippet: Human lung alveolar epithelial (A549) cells (CCL-185), Madin-Darby canine kidney (MDCK) cells (CCL-34), baby hamster kidney-21 (BHK-21) cells (CCL-10), and human embryonic 293T (HEK293T) cells (CRL-3216) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

Techniques: Infection, Quantitative RT-PCR, Transfection, Purification, Virus, Mutagenesis, Luciferase, Control, Knock-Out, Activation Assay, Binding Assay, Comparison

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) Schematic of luciferase assay: A549 cells were transfected with siRNAs and at 72 hpt (hours post-transfection), cells were infected with recombinant WSN virus expressing PB2-T2A-NanoLuc. After 48 hours, luciferase activity was measured (Figure S4C-D), providing read-out proportional to viral polymerase levels in infected, siRNA-treated cells . Created in BioRender https://BioRender.com/h91s731 (B) Log2-transformed relative luciferase units [Log2 (RLU)] normalised to a non-targeting siRNA control (siNT). Data are represented as mean ± standard deviation (SD) (n = 3 to 5 Statistical significances: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA and Dunnett’s multiple comparisons test, reference: siNT). See also Figure S4A-B.

Article Snippet: The human lung epithelial cell line A549 (86012804) and the human HEK293T (120220101) cell line were purchased from European Collection of Authenticated Cell Cultures (ECACC) and were grown under standard conditions (37 °C and 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11960044) supplemented with 10% fetal bovine serum (FBS) (Gibco, 0270106), GlutaMAX (Gibco 35050038), Sodium pyruvate (Gibco 11360039), penicillin–streptomycin (Gibco™ 15070063).

Techniques: Luciferase, Transfection, Infection, Recombinant, Virus, Expressing, Activity Assay, Transformation Assay, Control, Standard Deviation

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 of M2 and LAT1 (B) Log(RLU) normalised to siNT. Data are represented as mean ± SD (n = 3), with individual experiment means shown as circles. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. siSLC3A2 is replicated from . (C) Volcano plots showing fold-change (log2) versus significance (-log10 p-value) for SLC7A5 versus isotype control. Viral proteins are depicted in orange, SLC7A5 and SLC3A2 – in green. (D) Maximum intensity projection (MaxIP) of confocal images from A549 cells infected with WSN (Multiplicity of infection (MOI) 3) or mock at 12 hpi. Cells were stained for SLC7A5 (green), M2 (magenta), and nuclei (DAPI, cyan). Scale bars, 20 µm. (E) Pearson’s correlation coefficient quantification of SLC7A5 and M2 colocalisation in membrane regions. Analysis was performed in mock-infected cells (n = 64) and WSN-infected cells at 12 hpi (n = 119). Statistical significance was determined using the Mann-Whitney test (****p<0.0001). (F) Representative plane and intensity profile showing the co-localisation of SLC7A5 and M2 in A549 cells at 12 hpi.

Article Snippet: The human lung epithelial cell line A549 (86012804) and the human HEK293T (120220101) cell line were purchased from European Collection of Authenticated Cell Cultures (ECACC) and were grown under standard conditions (37 °C and 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11960044) supplemented with 10% fetal bovine serum (FBS) (Gibco, 0270106), GlutaMAX (Gibco 35050038), Sodium pyruvate (Gibco 11360039), penicillin–streptomycin (Gibco™ 15070063).

Techniques: Control, Infection, Staining, Membrane, MANN-WHITNEY

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 human lung epithelial cell line A549 (86012804) and the human HEK293T (120220101) cell line were purchased from European Collection of Authenticated Cell Cultures (ECACC) and were grown under standard conditions (37 °C and 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11960044) supplemented with 10% fetal bovine serum (FBS) (Gibco, 0270106), GlutaMAX (Gibco 35050038), Sodium pyruvate (Gibco 11360039), penicillin–streptomycin (Gibco™ 15070063).

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) Maximum projection of FISH confocal microscopy images of HEK293T cells transfected with either GFP (control), WSN viral proteins – NP, NS1, or a mini replicon vRNP system (including PA, PB1, PB2, NP and vRNA of NP), taken at 24 hpi. NEAT1_2 (paraspeckles) is shown in magenta, and GFP (control) or viral mRNAs (NP or NS1) are indicated in green, DNA is counterstained with DAPI (cyan). Scale bar: 20 µm. (B, C) CV of NEAT1_2 (B) and the number of paraspeckles per nucleus (C) across different conditions. Only GFP-positive cells were considered. (D) Maximum projection of confocal microscopy images of HEK293T cells transfected with either GFP (control) or co-transfected with PA-X and GFP, taken at 24 hpt. NEAT1_2 (paraspeckles) is shown in magenta, and GFP is indicated in green, DNA is counterstained with DAPI (grey). NEAT1_2 is used as a marker for paraspeckles to assess the impact of IAV proteins on paraspeckle integrity. (E,F) CV of NEAT1_2 in the nucleus and the number of paraspeckles per nucleus (F) across different conditions. Only GFP-positive cells were considered. (G) Confocal images showing the maximum projection of NEAT1_2 (magenta) in A549 cells treated with different concentrations of α-Amanitin for 20 h. DAPI (cyan) counterstains the nuclei. Scale bar = 20 μm. (H, I) CV of NEAT1_2 signal intensity (H) and the number of paraspeckles (I) per nucleus in A549 cells treated with α-Amanitin for 20 h. (J) Quantitative PCR analysis of NEAT1_2 and NEAT1_1 expression in A549 cells infected with WSN (MOI 3) normalised to GAPDH and to the mock-infected condition at each corresponding time point (n=3). (K) Quantitative PCR analysis of NEAT1_2 and NEAT1_1 expression in Calu-3 cells infected with WSN (MOI 3) normalised to GAPDH and to the mock-infected cells (n=3). (L) Western blot analysis of NONO protein levels in A549 cells infected with WSN (MOI 3) at different time points post-infection (3, 6, 9, 14, 20, and 24 hpi). Vinculin serves as the loading control. (M) Log₂ Label-Free Quantification (LFQ) intensity of paraspeckle proteins in A549 cells at 14 hpi with WSN (MOI=3), normalised to mock-infected cells. (B-E, G-J, M) 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 human lung epithelial cell line A549 (86012804) and the human HEK293T (120220101) cell line were purchased from European Collection of Authenticated Cell Cultures (ECACC) and were grown under standard conditions (37 °C and 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11960044) supplemented with 10% fetal bovine serum (FBS) (Gibco, 0270106), GlutaMAX (Gibco 35050038), Sodium pyruvate (Gibco 11360039), penicillin–streptomycin (Gibco™ 15070063).

Techniques: Confocal Microscopy, Transfection, Control, Marker, Real-time Polymerase Chain Reaction, Expressing, Infection, Western Blot

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 human lung epithelial cell line A549 (86012804) and the human HEK293T (120220101) cell line were purchased from European Collection of Authenticated Cell Cultures (ECACC) and were grown under standard conditions (37 °C and 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, 11960044) supplemented with 10% fetal bovine serum (FBS) (Gibco, 0270106), GlutaMAX (Gibco 35050038), Sodium pyruvate (Gibco 11360039), penicillin–streptomycin (Gibco™ 15070063).

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: Influenza virus recruits “PKC⍺-MEK1-ERK2” complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication

doi: 10.1101/2025.03.06.641832

Figure Lengend Snippet: (A) Plaque morphology and average plaque diameter of WT or recombinant NP mutant viruses. (B) Multicycle replication kinetics of the mutant NP viruses. Absolute (C) and relative (D) abundance of viral genome at different times of post infection were quantified using real time qRT-PCR. (E) Polymerase activity assay using negative sense viral RNA (vRNA) template along with WT and mutant NP reconstituted in HEK293T cells. (F) WT and mutant NP proteins expressed in A549 cells were fixed,immuno-stained with anti-NP antibody at 36 h.p.t and imaged using a confocal microscope. (G) A549 cells infected with WT or mutant viruses (MOI:5). At 2 and 8 hpi cells were fixed, immuno-stained using anti-RNP antibody and imaged using a confocal microscope. 50 cells from 5 different fields were analysed using image J software to present nuclear-cytoplasmic distributions of NP.

Article Snippet: Human lung epithelial cells (A549) (CCL-185), Human embryonic kidney (HEK) 293T cells (CRL-3216) and Madin Darby Canine Kidney (MDCK) (CCL-34) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO 2 along with antimycotic, penicillin and streptomycin antibiotics (Gibco).

Techniques: Recombinant, Mutagenesis, Infection, Quantitative RT-PCR, Activity Assay, Staining, Microscopy, Software

Journal: bioRxiv

Article Title: Influenza virus recruits “PKC⍺-MEK1-ERK2” complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication

doi: 10.1101/2025.03.06.641832

Figure Lengend Snippet: A549 cells were infected with WT and mutant viruses harboring phospho-null alanine substitutions at the ERK2 phosphorylation sites. Absolute (A) and relative (B) abundance of viral mRNA at different times of post infection were quantified using realtime qRT-PCR. (C) Ability of the WT and mutant NP proteins to support RNP activity and viral RNA synthesis using reporter RNPs with positive sense viral RNA (cRNA) template reconstituted in HEK293T cells through transient transfection. (D) Expression levels of the WT and mutant NP proteins assessed through western blot analysis.

Article Snippet: Human lung epithelial cells (A549) (CCL-185), Human embryonic kidney (HEK) 293T cells (CRL-3216) and Madin Darby Canine Kidney (MDCK) (CCL-34) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO 2 along with antimycotic, penicillin and streptomycin antibiotics (Gibco).

Techniques: Infection, Mutagenesis, Quantitative RT-PCR, Activity Assay, Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: Influenza virus recruits “PKC⍺-MEK1-ERK2” complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication

doi: 10.1101/2025.03.06.641832

Figure Lengend Snippet: (A) Influenza A/H1N1 NP was expressed in A549 cells through transient transfection, fixed and stained using specific antibody at 12, 24, 36 hours post transfection. Imaged using confocal microscope. 50 cells from 5 different filed was analysed using image J software to present nucelar-cytoplasmic distributions of NP. (B) WT and mutant NP proteins expressed in A549 cells were fixed and immuno-stained with anti-NP antibody at 12 and 24 hpt. Cells were imaged using confocal microscope.

Article Snippet: Human lung epithelial cells (A549) (CCL-185), Human embryonic kidney (HEK) 293T cells (CRL-3216) and Madin Darby Canine Kidney (MDCK) (CCL-34) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO 2 along with antimycotic, penicillin and streptomycin antibiotics (Gibco).

Techniques: Transfection, Staining, Microscopy, Software, Mutagenesis

Journal: bioRxiv

Article Title: Influenza virus recruits “PKC⍺-MEK1-ERK2” complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication

doi: 10.1101/2025.03.06.641832

Figure Lengend Snippet: A549 cells infected with WT A/WSN/1933 viruses (MOI:5). 2, 4 and 8 hpicells were fixed and immune-stained using anti-RNP antibody and imaged using confocal microscope. Nucleo-cytoplasmic distribution of NP was analysed using imageJ software.

Article Snippet: Human lung epithelial cells (A549) (CCL-185), Human embryonic kidney (HEK) 293T cells (CRL-3216) and Madin Darby Canine Kidney (MDCK) (CCL-34) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO 2 along with antimycotic, penicillin and streptomycin antibiotics (Gibco).

Techniques: Infection, Staining, Microscopy, Software

Journal: bioRxiv

Article Title: Influenza virus recruits “PKC⍺-MEK1-ERK2” complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication

doi: 10.1101/2025.03.06.641832

Figure Lengend Snippet: (A) Immunoprecipitation of NP from A/H1N1/WSN virus infected A549 cells (MOI-10) at 6 hpi using ANP antibody. (B) B/ Brisbane/ 60/ 2008 virus infected (MOI-10, 6 hpi) A549 cells subjected to immunoprecipitation using antiBNP antibody. A549 cells infected with influenza A/ H1N1 virus (MOI-5) were fixed at 2 and 8 hours of post infection and immunostained for NP and PKCα (C) or ERK (D) or MEK (E). Images were analysed with image J software to present subcellular distribution of the proteins (lower panel). (F) Super-resolution micrographs of the infected A549 cells showing precise co-localization of viral NP with PKCα, ERK and MEK at 2 and 8 hpi. Co-localization of PKC, ERK and MEK (in red line) with NP (in green line) was plotted through Image J software. (G) Schematic representation of proximity ligation assay. (H) Cells infected with influenza A/H1N1 virus or mock infected were subjected to PLA using anti-RNP (goat) and anti-ERK2 (rabbit) primary antibodies or with one of the primary antibody along with the IgG isotype control for the other (full panel shown in Figure S11). Post PLA, cells are stained with anti-NP antibody (mouse) and imaged. (I) Images were analysed for the PLA dots per cell using Image J software and plotted for quantitative representation. (J) Subcellular distribution of PLA dots and NP were analysed using image J software.

Article Snippet: Human lung epithelial cells (A549) (CCL-185), Human embryonic kidney (HEK) 293T cells (CRL-3216) and Madin Darby Canine Kidney (MDCK) (CCL-34) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO 2 along with antimycotic, penicillin and streptomycin antibiotics (Gibco).

Techniques: Immunoprecipitation, Virus, Infection, Software, Proximity Ligation Assay, Control, Staining

Journal: bioRxiv

Article Title: Influenza virus recruits “PKC⍺-MEK1-ERK2” complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication

doi: 10.1101/2025.03.06.641832

Figure Lengend Snippet: (A) Cells overexpressing PKCα-CAT-DN or control cells were infected with A/H1N1/WSN/1933 virus followed by fixing and immunostaining at indicated times of post infection. (B) Images from five different fields were analyzed to measure the extent of infection using image J software. (C) Virus titers from PKCα-CAT-DN overexpressing or control cells were measured using plaque assay at different times of post infections as indicated. (D) A549 cells were transduced with lentiviruses expressing PKCα specific or non-target (NTC) shRNAs. Knockdown efficiency was tested by western blot analysis and PKCα band intensity from three different experiments are plotted using image J software. PKCα deficient cells (PKCα-KD1, PKCα-KD2, PKCα-KD1+2) were infected with WT or NP-S450A/ S473A mutant viruses and titer was measured at 12 and 24 hpi. Relative (D-F) and absolute (G-I) virus titers for both the viruses at two different time points are plotted.

Article Snippet: Human lung epithelial cells (A549) (CCL-185), Human embryonic kidney (HEK) 293T cells (CRL-3216) and Madin Darby Canine Kidney (MDCK) (CCL-34) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C and 5% CO 2 along with antimycotic, penicillin and streptomycin antibiotics (Gibco).

Techniques: Control, Infection, Virus, Immunostaining, Software, Plaque Assay, Transduction, Expressing, Knockdown, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A and B ) The C-terminal domain of A. fumigatus HscA is required to inhibit phagosome maturation. ( A ) Immunostaining of Rab7-positive (Rab7 + ) phagosomes in A549 cells containing conidia from A. fumigatus wild-type (WT), Δ hscA , hscAΔC-myc , A. nidulans, and A. terreus . Extracellular fungal conidia were stained with calcofluor white (CFW). Dashed-line circles indicate internalized A. fumigatus WT conidia, and arrows mark the Rab7 + phagosomes. Scale bars, 10 μm. ( B ) Percentage of Rab7 + phagosomes containing conidia in A549 cells. Data represent the mean ± SD from three independent experiments. ( C and D ) The Y596 residue of A. fumigatus HscA compared to A. nidulans and A. terreus . ( C ) Schematic representation of HscA domains, including the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate-binding domain (SBD), which is further divided into the β-sandwich (SBD-β) and α-helical (SBD-α) subdomains. An alignment of the C-terminal 50 amino acid residues of HscA from A. fumigatus , A. nidulans , and A. terreus highlights differences at positions A592 and Y596 marked in yellow and green, respectively. Blue helices indicate α-helix structural elements based on the crystal structure of Chaetomium thermophilum CtSsb (ref. ). ( D ) Variation map of the C-terminal 50 residues of HscA from Eurotiomycetes fungi. ( E ) Graphical representation of hscA - myc gene, highlighting positions of amino acids 592 and 596. The enlarged section shows the alignment of DNA sequences from A. fumigatus Δ hscA strains expressing mutant hscA genes with point mutations at codons 592 and/or 596. ( F ) Western blot analysis of protein extracts from germlings of indicated A. fumigatus strains with antibodies against HscA, Myc, and GAPDH. ( G – I ) Y596 of A. fumigatus HscA is essential for regulating Rab7 and p11recruitment to phagosomes. ( G ) Rab7-positive phagosomes (Rab7 + PSs), ( H ) p11-positive phagocytic cups (p11 + PCs), and ( I ) p11 + PSs containing conidia were quantified. Data represent the mean ± SD with the number of independent experiments indicated at the bottom of each bar. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: Human lung epithelial cells A549 (Cat# 86012804-1VL, Sigma-Aldrich) were cultured in F-12K Nut Mix medium (Kaighn’s modification, Gibco) supplemented with 10% (v/v) artificial fetal calf serum (FCS) (HyClone FetalClone III serum, Cytiva) as described previously .

Techniques: Immunostaining, Staining, Residue, Binding Assay, Expressing, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: (A) Schematic showing the strategy used to detect the association or entry of recombinant proteins into live A549 cells. ( B – D ) Immunofluorescence staining of A549 cells incubated with recombinant proteins. (B) Immunostaining of A549 cells incubated with rHscA, rHsp70, or GFP for 2 hours at 4°C. rHscA and rHsp70 were detected indirectly using an antibody against the Strep-tag, while GFP was detected indirectly using an antibody against GFP. No recombinant protein or primary antibody were added to negative control. (C) Confocal microscopy images of A549 cells were used to ( D ) reconstruct cells as 3D surfaces. Cells were incubated with rHscA and stained with an anti-CD9 antibody. rHscA was indirectly detected using an antibody against the Strep-tag. In the reconstructed 3D images, membrane-associated rHscA was labeled in red, while internalized rHscA was labeled in yellow. Arrows indicate rHscA proteins crossing the plasma membrane. See also Video S1.

Article Snippet: Human lung epithelial cells A549 (Cat# 86012804-1VL, Sigma-Aldrich) were cultured in F-12K Nut Mix medium (Kaighn’s modification, Gibco) supplemented with 10% (v/v) artificial fetal calf serum (FCS) (HyClone FetalClone III serum, Cytiva) as described previously .

Techniques: Recombinant, Immunofluorescence, Staining, Incubation, Immunostaining, Strep-tag, Negative Control, Confocal Microscopy, Membrane, Labeling

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) The C-terminus of HscA is exposed on the surface of phagosomes. ( A ) Immunostaining of phagosomes containing conidia of hscA-myc or hscA L -myc strains isolated from A549 cells. Scale bars, 2 μm. ( B ) Relative signal intensities of the respective emission fluorescence along the lines drawn across the phagosomes shown in ( A ). ( C ) Quantification of exposure distance from a representative experiment. Data represent the mean ± SD; n = 16 individual phagosomes were analyzed. ( D and E ) Biotinylation of host cell proteins by HscA-miniTurboID (HscA-mT). ( D ) A549 cells incubated with conidia of strains hscA-mT or mT - hscA were stained with streptavidin and an antibody against RAB7. ( E ) Phagosomes with positive streptavidin (Strep + ) signal were quantified. The number of independent experiments is indicated at the bottom of the bars. ( F and G ) GAL3 is recruited to damaged phagosomes containing A. fumigatus conidia in A549 cells after 8 hours of infection. ( F ) A549 cells incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. ( G ) Quantification of GAL3 + phagosomes containing conidia in A549 cells. ( H and I ) Phagosomal SYTOX is released to the nucleus upon damage of the phagosome membrane. ( H ) Representative A549 cells whose lysosomes were loaded with SYTOX-Green, were incubated with A. fumigatus for 16 h. CellMask and CFW were used to stain the cell membrane and A. fumigatus cell wall, respectively. ( I ) Cells with SYTOX signal in the nuclei were quantified. ( J – L ) HscA-dependent phagosomal damage in hMDMs. ( J ) hMDMs incubated with A. fumigatus conidia were immunostained with indicated antibodies. Scale bars, 5 μm. DIC, differential interference contrast. ( K ) GAL3 + phagosomes and ( L ) RAB7 + phagosomes in hMDMs were quantified. Statistics: Error bars represent the mean ± SD. For C and E, p -values were calculated using unpaired two-tailed t test. For G, I, K, and L, p -values are calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Gray dots represent the calculated values from individual microscopic images (G, n = 42–47; I, n = 25–28; K and L, n = 46–54), and colored dots represent the summarized result of individual experiments ( n = 3).

Article Snippet: Human lung epithelial cells A549 (Cat# 86012804-1VL, Sigma-Aldrich) were cultured in F-12K Nut Mix medium (Kaighn’s modification, Gibco) supplemented with 10% (v/v) artificial fetal calf serum (FCS) (HyClone FetalClone III serum, Cytiva) as described previously .

Techniques: Immunostaining, Isolation, Fluorescence, Incubation, Staining, Infection, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: Human lung epithelial cells A549 (Cat# 86012804-1VL, Sigma-Aldrich) were cultured in F-12K Nut Mix medium (Kaighn’s modification, Gibco) supplemented with 10% (v/v) artificial fetal calf serum (FCS) (HyClone FetalClone III serum, Cytiva) as described previously .

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – C ) Deletion of fungal HscA or human host p11 gene, or chelation of Ca 2+ , increased phagosome maturation. ( A ) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated phagosomal markers: RAB7 and ANXA2 on the top row, and GAL3 and LAMP1 on the bottom row. Yellow arrows label phagosomes positive for both markers, while white arrows mark phagosomes positive for a single marker. Scale bars, 5 μm. ( B and C ) Phagosomes positive for ( B ) RAB7 and ( C ) LAMP1 were quantified. ( D and E ) Activation of TFEB by p11 deletion or Ca 2+ chelation. ( D ) Immunostaining of A549 cells infected with WT conidia. Dashed-line circles indicate the regions of nuclei. Scale bars, 10 μm. ( E ) Cells with TFEB localized in the nuclei were quantified. Statistics: Error bars represent the mean ± SD; p values were determined using one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments for figures B and C is indicated at the base of each bar. For E, grey dots represent the calculated values from individual microscopic images ( n = 41–68) and colored dots represent summarized results from individual experiments ( n = 3 for BAPTA-AM-treated cells and n = 4 for untreated cells).

Article Snippet: Human lung epithelial cells A549 (Cat# 86012804-1VL, Sigma-Aldrich) were cultured in F-12K Nut Mix medium (Kaighn’s modification, Gibco) supplemented with 10% (v/v) artificial fetal calf serum (FCS) (HyClone FetalClone III serum, Cytiva) as described previously .

Techniques: Immunostaining, Incubation, Marker, Activation Assay, Infection