Immortalized cell lines<\/p>\n
Human A549 (86012804; ECACC) and HEK293T (CRL-3216; ATCC) cells were cultured in DMEM (11960044; Gibco) supplemented with 10% FBS (0270106; Gibco), GlutaMAX (35050038; Gibco), sodium pyruvate (11360039; Gibco) and penicillin\u2013streptomycin (15070063; Gibco) at 37\u2009\u00b0C and 5% CO2. Calu-3 cells (no catalogue number available\u2014provided by Petr Chlanda, Heidelberg University; originally from Prof. Ralf Bartenschlager, Heidelberg University) were maintained in the same medium with 20% FBS. MDCK (84121903; ECACC), MDCK.II (00062107; ECACC), MDCK-SIAT1 (05071502-1VL; Sigma-Aldrich) and MDBK (no catalogue number available\u2014provided by Ervin Fodor, University of Oxford; Sir William Dunn School of Pathology Cell Bank stock) cells were grown in MEM supplemented with 10% FBS, GlutaMAX, sodium pyruvate and penicillin\u2013streptomycin. MEFs (no catalogue number available\u2014provided by Prof. Dr. Michael Bader, Max Delbr\u00fcck Center) were cultured in DMEM with 10% FBS, GlutaMAX, sodium pyruvate, penicillin\u2013streptomycin and non-essential amino acids (11140050; Gibco). HeLa (Kyoto; no catalogue number is available for this line, provided by the Mahamid laboratory, which had received them from the Sara Cuylen-Haering laboratory, originally from Daniel Gerlich, Institute of Molecular Biotechnology of the Austrian Academy of Sciences; the Gerlich laboratory reported this HeLa Kyoto stock as originally obtained from S. Narumiya, Kyoto University, RRID:CVCL_1922<\/a>, authenticated by multiplex human cell line authentication testing) were cultured in DMEM (11960044; Gibco) supplemented with 10% FBS (0270106; Gibco), GlutaMAX (35050038; Gibco), sodium pyruvate (11360039; Gibco) and penicillin\u2013streptomycin (15070063; Gibco) at 37\u2009\u00b0C and 5% CO2. A549 and HEK293T cells were authenticated by short tandem repeat profiling. All cell lines were routinely tested for mycoplasma contamination and confirmed negative.<\/p>\n Primary cells<\/p>\n Primary HBEpCs (C-12640, donor lot number 499Z012.1; Promocell) were maintained in Airway Epithelial Cell Growth Medium (C-21060; Promocell) and cultured according to the manufacturer\u2019s instructions in Airway Epithelial Cell Growth Medium at 37\u2009\u00b0C with 5% CO2. HBEpCs were maintained as undifferentiated submerged cultures and were not differentiated at an air\u2013liquid interface. Primary cells were used at passages 2\u20136 for all experiments. All cell lines were routinely tested for mycoplasma contamination (MycoplasmaCheck; Eurofins).<\/p>\n Virus production<\/p>\n WSN was produced in confluent MDBK cells infected at a multiplicity of infection (MOI) of 0.001 in DMEM containing 0.5% FBS. After 36\u2009hours incubation at 37\u2009\u00b0C and 5% CO2, when full cytopathic effect was observed, supernatants were collected, clarified by centrifugation (1,500g, 10\u2009minutes, 4\u2009\u00b0C) and aliquoted for storage at \u221280\u2009\u00b0C. pdm09 and H3N2 were produced in MDCK cells infected at an MOI of 0.001 in serum-free DMEM supplemented with 0.2% BSA and 1\u2009\u00b5g\u2009ml\u22121 TPCK-treated trypsin. After 36\u201348\u2009hours incubation at 37\u2009\u00b0C and 5% CO2 and confirmation of full cytopathic effect and successful HA assay, viral supernatants were clarified and stored as mentioned earlier.<\/p>\n Recombinant SC35M (H7N7) was generated by transfecting HEK293T cells with 8 plasmids (pHW2000) encoding all 8 influenza gene segments (1\u2009\u03bcg each) and Lipofectamine 2000 (Invitrogen) according to the manufacturers\u2019 protocol. After an incubation period of 6\u2009hours, cell culture medium was removed and replaced with fresh DMEM. At 48\u2009hours post-transfection, supernatants were collected to inoculate fresh MDCK. For a secondary infection, supernatants from previously infected MDCK cells were collected at 48\u2009hours post-infection, stored at \u221270\u2009\u00b0C and used for inoculation of fresh MDCK cells. After clear cytopathic effect development, supernatants were collected and processed as described earlier.<\/p>\n Virus infection<\/p>\n Cells were infected with IAV strains A\/WSN\/33 (H1N1), A\/California\/07\/2009 (pdm09, H1N1), A\/Aichi\/2\/1968 (H3N2) or SC35M (mouse-adapted variant of the highly pathogenic avian influenza virus strain SC35 derived from A\/Seal\/Massachusetts\/1\/80 (H7N7)) at the indicated MOI. Before infection, virus inocula were diluted in infection medium consisting of serum-free DMEM supplemented with 0.2% BSA and antibiotics. Cells were washed once with PBS and incubated with the virus inoculum for 30\u2009minutes at 4\u2009\u00b0C, followed by an additional 30\u2009minutes at 37\u2009\u00b0C with gentle rocking to allow virus attachment and entry. For SC35M, cells were incubated with virus inoculum for 30\u2009minutes at 37\u2009\u00b0C with gentle shaking to allow even distribution of virus particles on the cell layer. After infection, the inoculum was removed and replaced with fresh infection medium.<\/p>\n For infections with A\/WSN\/33 and SC35M, infection medium did not contain trypsin. For infections with A\/California\/07\/2009 and A\/Aichi\/2\/1968, infection medium was supplemented with TPCK-treated trypsin (1\u2009\u00b5g\u2009ml\u22121) as required for these strains to support multicycle replication. Infected cells were incubated at 37\u2009\u00b0C and 5% CO2 and collected at the indicated time points for downstream analyses.<\/p>\n Reconstitution of recombinant IAV overexpressing NanoLuc on PB2<\/p>\n A recombinant IAV expressing NanoLuc fused to PB2 was generated using a 12-plasmid reverse genetics system as described previously34<\/a>. The system included eight plasmids encoding the vRNA segments and four expression plasmids for PB2, PB1, PA and NP under control of a human Pol I promoter. The NanoLuc reporter gene was inserted in-frame at the amino terminus of PB2, separated by a self-cleaving T2A peptide to allow independent translation.<\/p>\n HA assay<\/p>\n The HA assay was performed in 96-well round-bottom plates using 2-fold serial dilutions of virus in PBS (50\u2009\u00b5l per well). An equal volume (50\u2009\u00b5l) of 0.5% chicken erythrocyte suspension was added to each well. Plates were incubated for 30\u2009minutes at 4\u2009\u00b0C, followed by 30\u2009minutes at room temperature. HA titres were determined visually as the highest virus dilution producing complete haemagglutination, defined by a uniform lattice across the well. Results are expressed in haemagglutination units.<\/p>\n Plaque assay<\/p>\n For WSN, confluent MDCK cells in 6-well plates were infected with 330\u2009\u00b5l of serially diluted virus in infection media (DMEM with 0.2% BSA). Plates were incubated for 30\u2009minutes at 4\u2009\u00b0C, followed by 30\u2009minutes at 37\u2009\u00b0C with gentle rocking. Cells were overlaid with Avicel-containing media (DMEM, 0.2% BSA) and incubated for 72\u2009hours. After removing the overlay, cells were washed with PBS and stained with 1% crystal violet for 5\u2009minutes. Plaques were counted to calculate viral titre in plaque-forming units per millilitre (PFU\u2009ml\u22121). For pdm09 and H3N2, MDCK-II cells were infected as above and overlaid with DMEM containing Avicel, 0.2% BSA and 1\u2009\u00b5g\u2009ml\u22121 TPCK-treated trypsin. After 72\u2009hours, cells were fixed with 4% paraformaldehyde for 30\u2009minutes at 4\u2009\u00b0C and blocked with 0.3% hydrogen peroxide. Viral plaques were detected by immunostaining with anti-NP antibodies (Aichi, MA5-42364, Thermo Fisher; pdm09, ab43821, Abcam; 1:1,000) for 1\u2009hour at room temperature, followed by HRP-conjugated secondary antibody and KPL TrueBlue substrate (15\u2009minutes). Viral titres were calculated as PFU\u2009ml\u22121:<\/p>\n $$\\mathrm{Titre}\\,\\left(\\frac{\\mathrm{PFU}}{\\mathrm{ml}}\\right)=\\frac{\\left(\\mathrm{number}\\,\\mathrm{of}\\,\\mathrm{plaques}\\,\\times \\,\\mathrm{dilution}\\,\\mathrm{factor}\\right)}{\\mathrm{volume}\\,\\mathrm{of}\\,\\mathrm{virus}\\,\\mathrm{innoculum}\\,(\\mathrm{ml})}$$<\/p>\n Luciferase assay<\/p>\n A549 cells were seeded at 1\u2009\u00d7\u2009105 cells per well in 24-well plates and transfected 12\u2009hours later in duplicate with 10\u2009\u00b5M siRNAs using RNAiMAX (Thermo Fisher Scientific), according to the manufacturer\u2019s instructions. After 72\u2009hours incubation at 37\u2009\u00b0C and 5% CO2, cells were washed with infection media and infected with PB2-T2A-NanoLuc recombinant WSN at an MOI of 0.01. At 48\u2009hours post-infection, cells were lysed in 200\u2009\u00b5l Passive Lysis Buffer (Promega) per well and incubated at room temperature for 20\u201330\u2009minutes on a rocking platform. For luciferase activity, 25\u2009\u00b5l of lysate was mixed with 25\u2009\u00b5l NanoGlo substrate (Promega) in a white 96-well plate. Luminescence was measured after 5\u201310\u2009minutes equilibration using a luminometer (1,000\u2009ms integration time).<\/p>\n Viral growth kinetics<\/p>\n For viral growth kinetics, cells were infected at low multiplicity to allow multicycle replication. A549 cells and HBEpCs were seeded at 2\u2009\u00d7\u2009105 cells per well, transfected with the indicated siRNAs for 72\u2009hours, and subsequently infected with WSN at an MOI of 0.001 or pdm09 at an MOI of 0.01. For experiments performed in KO or overexpressed cell lines, cells were seeded at 7.5\u2009\u00d7\u2009105 cells per well before infection. Following infection, cells were washed and maintained in the appropriate infection medium.<\/p>\n At the indicated time points post-infection, 50\u2009\u00b5l of supernatant was collected and stored at \u221270\u2009\u00b0C until analysis. Viral titres were determined by plaque assay on MDCK cells (WSN) or MDCK-SIAT1 cells (pdm09) as described earlier. Growth curves are presented as PFU\u2009ml\u22121 at 24\u2009hpi, 48\u2009hpi and 72\u2009hpi.<\/p>\n FISH<\/p>\n Cells (1\u2009\u00d7\u2009105 per well) were seeded onto sterilized glass coverslips in 24-well plates and grown to ~70\u201380% confluence. Cells were washed with DPBS, fixed in 4% paraformaldehyde for 10\u2009minutes at room temperature and permeabilized in 70% ethanol overnight at \u221220\u2009\u00b0C. Hybridization and amplification were performed using the hybridization chain reaction (HCR v.3.0) protocol adapted from ref. 79<\/a>, with optimizations for detecting IAV vRNA, mRNA and antisense RNA (see Supplementary Table 5<\/a> for probe sequences and design). Briefly, fixed cells were prehybridized with Probe Hybridization Buffer (Molecular Instruments) and incubated overnight with 2\u2009pmol of each probe at 37\u2009\u00b0C. Following washes in Probe Wash Buffer and SSCT, amplification was carried out overnight at room temperature using snap-cooled HCR hairpins (h1 and h2). Cells were washed in 5\u00d7 SSCT, stained with DAPI and mounted using Dako antifade mountant (Agilent). Samples were imaged using a confocal microscope with appropriate filter settings for DAPI and HCR fluorophores. Acquisition settings were optimized to reduce background and enhance signal detection.<\/p>\n siRNA transfection<\/p>\n All siRNAs were purchased from Qiagen (Supplementary Table 6<\/a>). A549 cells at ~60% confluence were transfected with 5\u2009pmol siRNA using Lipofectamine RNAiMAX (13778075; Thermo Fisher Scientific) according to the manufacturer\u2019s protocol. Knockdown efficiency was assessed 72\u2009hours post-transfection by qPCR or western blot.<\/p>\n PLA<\/p>\n PLA was performed using the Duolink In Situ PLA kit (Sigma-Aldrich) following the Sigma protocol. A549 cells were seeded on glass coverslips and infected as described earlier. At the indicated time point post-infection, cells were washed with PBS, fixed with 4% paraformaldehyde for 10\u2009minutes at room temperature and permeabilized with PBS containing 0.2% Triton X-100 for 10\u2009minutes at room temperature, according to the manufacturer\u2019s instructions. Next, cells were blocked in the supplied blocking solution and incubated with the primary antibodies. After washing, PLA probes were applied, followed by ligation and rolling-circle amplification steps. PLA signals were detected using Duolink detection reagents coupled to Alexa Fluor 488. Nuclei were counterstained with DAPI, and coverslips were mounted using Dako mounting medium (Agilent). Samples were imaged by fluorescence microscopy using identical acquisition settings across conditions, and PLA puncta and signal intensity were quantified from maximum z-projection images.<\/p>\n The following primary antibodies were used for PLA: anti-IAV nucleoprotein (NP) (clone C43; Abcam), anti-NS1 (EPR28247-51; Abcam), anti-HA (clone 1.B.408; Abcam), anti-PB2 (PA5-32220; Thermo Fisher Scientific), anti-M1 (clone GA2B, MA1-80736; Thermo Fisher Scientific), anti-hnRNPK (F45P9C7; Thermo Fisher Scientific), anti-RAB11A (67902-1-Ig; Proteintech), anti-hnRNPA1 (67844-1-Ig; Proteintech), anti-NF45 (67685-1-Ig; Proteintech), anti-hnRNPA2B1 (67445-1-Ig; Proteintech), anti-CCAR2 (66497-1-Ig; Proteintech), anti-SFPQ (67129-1-Ig; Proteintech), anti-nucleolin (D4C7O, number 14574; Cell Signaling Technology), anti-B4GALT1 (PA5-52744; Thermo Fisher Scientific), anti-VIP36 (PA5-90437; Thermo Fisher Scientific), anti-hnRNPM (26897-1-Ig; Proteintech), anti-ERP29 (24344-1-AP; Proteintech), anti-GLUT1 (21829-1-AP; Proteintech), anti-RPL35 (14826-1-AP; Proteintech), anti-ERGIC-53 (13364-1-AP: Proteintech), anti-hnRNPC (11760-1-AP; Proteintech) and anti-ANP32B (10843-1-AP; Proteintech).<\/p>\n Cell viability<\/p>\n Cell viability was measured using the CellTiter-Glo 2.0 Assay Kit (G9241; Promega). Treated cells in 24-well plates were incubated with reagent directly added to the culture medium, followed by 10\u2009minutes of shaking at room temperature. Luminescence, proportional to intracellular ATP levels, was measured using a plate reader and normalized to untreated controls.<\/p>\n CRISPR\u2013Cas9-mediated KO cell lines<\/p>\n Single guide RNAs were designed using Benchling or purchased as validated guides (IDT) and resuspended at 1\u2009\u00b5M. Recombinant Cas9 protein (EMBL Protein Production Facility) was diluted to 1\u2009\u00b5M in Opti-MEM. A549 cells were seeded at 1.5\u2009\u00d7\u2009105 cells per well in 6-well plates at <50% confluence to ensure high KO efficiency. Cas9\u2013single guide RNA ribonucleoprotein (RNP) complexes were formed in Opti-MEM and transfected using Lipofectamine RNAiMAX. After 96\u2009hours, cells were either collected for genomic DNA (NEB kit) or seeded at 0.5 cells per well in 96-well plates for clonal selection. Target loci were PCR-amplified, gel-purified and sequenced (Sanger). Indel frequency was analysed using TIDE, and KO was confirmed by western blotting.<\/p>\n Plasmid transfection<\/p>\n Plasmid transfections were performed in HEK293T cells at 70\u201380% confluence using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer\u2019s instructions. Plasmid DNA and Lipofectamine 3000 reagent were each diluted in Opti-MEM (Thermo Fisher Scientific), combined and incubated for 15\u2009minutes at room temperature to allow DNA\u2013lipid complex formation. The complexes were added dropwise to the cells and incubated for 4\u20136\u2009hours at 37\u2009\u00b0C in 5% CO2, after which the medium was replaced with fresh complete DMEM. Cells were incubated for an additional 16\u201348\u2009hours before analysis. Transfection efficiency was assessed by cotransfection with a GFP-expressing plasmid or by fluorescence microscopy following immunofluorescence or FISH staining.<\/p>\n Lentiviral cell line generation<\/p>\n Lentiviral vectors encoding mEGFP-tagged NONO, PSPC1 or SFPQ were cotransfected into HEK293T cells with packaging plasmids using a calcium phosphate transfection protocol. Supernatants containing lentiviral particles were collected after 48\u2009hours, filtered and stored at \u221270\u2009\u00b0C. A549 cells were transduced with viral supernatants in the presence of polybrene and incubated for 48\u201372\u2009hours to allow stable genomic integration. Transduced cells were selected using 10\u2009\u00b5g\u2009ml\u22121 Blasticidin and maintained in complete DMEM. Expression of tagged proteins was confirmed by fluorescence microscopy and western blotting. Protein functionality was verified by their colocalization with NEAT1 in paraspeckles.<\/p>\n RNA extraction, reverse transcription and qPCR<\/p>\n Total RNA was extracted using the RNeasy Kit (Qiagen) according to the manufacturer\u2019s instructions. Cells were lysed in buffer RLT supplemented with 5\u2009mM TCEP, and RNA was purified on RNeasy columns with RW1 and RPE wash buffers. RNA was eluted in RNase-free water, quantified using a NanoDrop spectrophotometer and stored at \u221270\u2009\u00b0C.<\/p>\n Reverse transcription was performed with up to 2\u2009\u00b5g RNA per 10\u2009\u00b5l reaction using the High-Capacity cDNA Reverse Transcription Kit (4368814; Thermo Fisher Scientific). qPCR was carried out using PowerUp SYBR Green Master Mix (A25742; Thermo Fisher Scientific) with gene-specific primers (Supplementary Table 7<\/a>). Thermal cycling and melt curve analysis were performed according to the manufacturer\u2019s protocol. Relative gene expression was quantified using the \u0394\u0394Ct method and normalized to GAPDH.<\/p>\n Immunoblotting<\/p>\n Whole-cell lysates were resolved by SDS\u2013PAGE using 4\u201312% precast TG PRiME gels (SERVA) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in 2.5% BSA (in TBS with 0.01% Tween-20) for 1\u2009hour at room temperature, followed by overnight incubation at 4\u2009\u00b0C with primary antibodies diluted in blocking buffer. After 3 washes in TBS-Tween, membranes were incubated for 1\u2009hour at room temperature with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5,000), washed again and developed using enhanced chemiluminescence (A38555; Thermo Fisher Scientific). Antibodies used for immunoblotting are listed in Supplementary Table 8<\/a>. Uncropped blots are available in the Source data<\/a>.<\/p>\n Affinity pulldown of Twin-Strep-tagged proteins<\/p>\n HEK293T cells were transfected with Twin-Strep-tagged constructs (NONO, M2 or empty vector control) using PEI at a 1:3 DNA:PEI ratio. At 24\u2009hours post-transfection, cells were collected by scraping in PBS, washed and lysed in IP lysis buffer (Thermo Fisher) supplemented with HALT protease inhibitor cocktail. Lysates were clarified (17,000g, 20\u2009minutes, 4\u2009\u00b0C) and incubated with Strep-Tactin magnetic beads (IBA Lifesciences) pre-equilibrated in Buffer W. After 1\u2009hour incubation at room temperature with rotation, beads were washed and bound proteins were eluted in Laemmli buffer at 95\u2009\u00b0C for 2\u2009minutes. Eluates were analysed by SDS\u2013PAGE and western blot.<\/p>\n IP<\/p>\n A549 cells were seeded in 10\u2009cm dishes (4\u2009\u00d7\u2009106 cells per dish) and infected with WSN (MOI 3) for 14\u2009hours. Cells were collected and lysed in IP buffer (Thermo Fisher) supplemented with HALT protease inhibitor (Thermo Fisher). For certain IPs (for example, NP, NONO, SFPQ and PSPC1), 0.1% SDS was added to reduce non-specific binding. Lysates were cleared by centrifugation (max speed, 20\u2009minutes, 4\u2009\u00b0C). IP was performed with 10\u2009\u00b5g of antibody and 500\u2009\u00b5l of cleared lysate for 4\u2009hours at 4\u2009\u00b0C, followed by incubation with Protein A\/G or G Dynabeads (Thermo Fisher) for 1\u2009hour at room temperature. Isotype-matched control antibodies were used in all experiments. Beads were washed with TBS\u2009+\u20090.05% Tween-20 and lysis buffer and finally with TBS alone. For AP\u2013MS, bound proteins were eluted in 8\u2009M guanidine HCl at 95\u2009\u00b0C and precipitated in cold ethanol overnight. Inputs were processed by methanol\u2013chloroform precipitation. For western blotting, elution was performed with 0.2\u2009M glycine (pH 2.0), followed by Tris neutralization and\/or SDS loading buffer. Eluates were analysed by SDS\u2013PAGE, western blot or mass spectrometry as described later. All AP\u2013MS experiments were performed in triplicate.<\/p>\n SHVIP sample preparation<\/p>\n Cells were infected at an MOI of 5 and, at 5\u2009hpi, incubated in labelling medium containing 500\u2009\u00b5M L-homopropargylglycine (L-HPG) in methionine-free DMEM with 0.2% BSA. At 14\u2009hpi, cells were washed with PBS and collected by scraping. For cross-linking, cell pellets were resuspended in PBS and cross-linked with 5\u2009mM DSSO for 1\u2009hour at room temperature with constant agitation. The reaction was quenched with 30\u2009mM Tris (pH 8.0) for 20\u2009minutes. Cells were lysed in buffer containing 200\u2009mM Tris (pH 8.0), 4% CHAPS, 1\u2009M NaCl, 8\u2009M urea and protease inhibitors. Lysates were frozen at \u221280\u2009\u00b0C until enrichment. After thawing, lysates were treated with Benzonase and sonicated (10\u2009\u00d7\u200930\u2009second on\/off cycles; Bioruptor Pico) and then clarified by centrifugation (10,000g, 10\u2009minutes). A 50\u2009\u00b5l aliquot was saved as input. Enrichment of HPG-labelled proteins was performed as described previously80<\/a>: 800\u2009\u00b5l of lysate was incubated with 200\u2009\u00b5l picolyl-azide agarose beads (Click Chemistry Tools), and copper(I)-catalysed click chemistry was performed overnight at room temperature with rotation. Proteins were reduced with 10\u2009mM DTT (70\u2009\u00b0C, 15\u2009minutes) and alkylated with 40\u2009mM chloroacetamide (room temperature in the dark). Beads were washed sequentially in gravity columns with: 1% SDS, 250\u2009mM NaCl and 5\u2009mM EDTA in 100\u2009mM Tris (pH 8.0); 8\u2009M urea in 100\u2009mM Tris (pH 8.0); 80% acetonitrile (ACN) in water; 5% ACN in 50\u2009mM triethylammonium bicarbonate (TEAB); and 5% ACN with 2\u2009M urea in 50\u2009mM TEAB. Bound proteins were digested on-bead overnight at 37\u2009\u00b0C with trypsin and LysC in the final wash buffer. Peptides were collected, acidified with 1% formic acid, desalted using C18 stage tip (or Sep-Pak C8 columns for cross-linked peptides), dried by vacuum centrifugation and stored at \u221220\u2009\u00b0C until LC\u2013MS or further processing. Input samples were processed by methanol\u2013chloroform precipitation and digested in buffer containing 1% sodium deoxycholate, 5\u2009mM TCEP and 40\u2009mM 2-chloroacetamide in 50\u2009mM TEAB. The resulting peptides were desalted as described earlier.<\/p>\n Off-line fractionation of cross-linked peptide samples<\/p>\n To enrich cross-linked peptides, HPG-enriched samples were first subjected to strong cation exchange (SCX) chromatography using a polysulfethyl A column. Peptides were separated with a 95-minute linear gradient and collected in 45-second intervals. Fractions were desalted, dried and stored at \u221220\u2009\u00b0C. After initial measurement, selected SCX fractions were pooled and further separated by size exclusion chromatography (SEC) using a Superdex 30 column. Fractions were collected every 2\u2009minutes, dried and stored at \u221220\u2009\u00b0C before LC\u2013MS\/MS.<\/p>\n LC\u2013MS\/MS of bottom-up proteomics and AP\u2013MS samples<\/p>\n Bottom-up samples and AP\u2013MS eluates were analysed using Orbitrap Fusion Tribrid or Exploris 480 instruments coupled to nano-LC systems. Peptides were loaded onto a 50\u2009cm in-house packed C18 column and separated by 120\u2013180\u2009minute gradients.<\/p>\n For bottom-up and AP\u2013MS samples, MS1 scans were acquired at 120,000 resolution, dynamic exclusion was set to 40\u2009seconds, precursors (charge +2 to +4) were isolated (1.6\u2009m\/z window) and fragmented by higher-energy collisional dissociation (HCD) at 30% normalized collision energy (NCE), with MS2 detection performed in the ion trap (Fusion) or Orbitrap (Exploris).<\/p>\n LC\u2013MS\/MS of cross-linked samples<\/p>\n Cross-linked peptides were analysed on an Orbitrap Fusion Lumos equipped with a FAIMS Pro Duo interface. MS1 scans were performed at 120,000 resolution using FAIMS voltages (\u221250\u2009V, \u221260\u2009V and \u221275\u2009V). Precursors (charge +4 to +8) were fragmented by stepped HCD (21%, 27% and 33% NCE) and MS2 scans acquired in the Orbitrap at 60,000 resolution.<\/p>\n Bottom-up proteomics data analysis<\/p>\n Raw files were searched using MaxQuant v.1.6.2.6a81<\/a> against the human SwissProt database (release 2020) and the IAV\/WSN\/1933 (H1N1) proteome. Trypsin was used for in silico digestion (max 2 missed cleavages). Carbamidomethylation (C) was set as fixed modification and variable modifications, including oxidation (M), acetylation (N-term) and methionine replacement with HPG, where appropriate. iBAQ and label-free quantification (LFQ) quantification were enabled. Contaminants, reverse hits and proteins identified only by site were removed. For LFQ, missing values were imputed as described previously16<\/a>. Fold changes were log2-transformed and P values were computed using 2-sided t tests. All AP\u2013MS experiments were performed in triplicates.<\/p>\n Raw data were searched using Scout (v.1.5.1)82<\/a> with DSSO cross-linker settings and residue specificity for K, S, T and Y. Search parameters included: minimum peptide length, 6; max missed cleavages, 3; precursor mass tolerance, 10\u2009ppm; fragment mass tolerance, 20\u2009ppm; static mod, carbamidomethylation (C); and variable mod, oxidation (M).<\/p>\n Data were filtered at 2% or 5% FDR on cross-link spectrum match, residue-pair and PPI levels. For performance evaluation (Extended Data Fig. 1m\u2013r<\/a>), SCX fractions were searched per replicate. For the full dataset, SCX and SEC files were combined for global analysis.<\/p>\n Glycosylation analysis<\/p>\n For each condition, 5 million A549 cells were seeded in a 15\u2009cm dish. After 12\u2009hours, cells were transfected with siRNAs targeting B4GALT1 or LMAN2 (2 independent siRNAs per target) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer\u2019s instructions. At 72\u2009hours post-transfection, cells were infected with WSN at an MOI of 3 as described earlier. At 14\u2009hpi, cells were collected by scraping, washed with PBS and frozen.<\/p>\n Glycoproteomics experiments were performed as described previously48<\/a> with minor adjustments: after lysis, digestion and glyco-enrichment, the samples were labelled with tandem mass tag (TMT) reagents and pooled. The samples were prefractionated into 24 fractions. After analysis of the glycopeptide samples on a Exploris 480, generated raw files were searched using MSFragger v.4.0 in Fragpipe v.20.0 against the Swissprot Homo sapiens database (UP000005640, 20,443 entries) combined with the influenza A database (UP000009255, 13 entries).<\/p>\n For the quantitative analysis of proteomics and glycoproteomics data, we first normalized the TMT reporter intensities for glycopeptides and proteins with complete quantification across all conditions (that is, TMT reporter ion intensity different from zero for all channels) using the normalizeVSN function (variance-stabilizing normalization; VSN) of the limma R package. We corrected glycopeptide intensities per protein by removing abundance-derived intensity using a linear regression for all glycopeptides with a matched total protein intensity. The corrected and normalized glycopeptide intensities and the normalized protein intensities were used for the differential expression analysis using the limma R package. For the design of the differential expression analysis, we considered the knockdown condition and the sample replicate. Contrasts were set for the comparison between the non-targeting control and the respective knockdown conditions.<\/p>\n Glycosylation at the N27\/N28 sites of HA could not be assessed, as the corresponding amino-terminal region (residues 3\u201343) lacks lysine and arginine residues and therefore does not generate detectable peptides under the trypsin-based proteomics and glycoproteomics workflows used in this study.<\/p>\n Confocal microscopy<\/p>\n All imaging was conducted using a Nikon Ti2 microscope equipped with a CSU-W1 spinning disk confocal unit and an Andor DU-888 X-11633 camera. The microscope was controlled by NIS-Elements software and used a 100\u00d7\/1.49 SR Apo TIRF AC oil immersion objective (Nikon). For live-cell imaging, cells were seeded in Ibidi \u03bc-Slide 4-well chambers (NC0685967; Ibidi) and maintained at 37\u2009\u00b0C with 5% CO2. Imaging was performed using the 405\u2009nm (DAPI channel), 488\u2009nm (GFP channel) and 561\u2009nm (Cy5 channel) lasers, with exposure times optimized for each experiment. The camera was maintained at \u221269.4\u2009\u00b0C, with a binning of 1\u2009\u00d7\u20091 and a readout speed of 30\u2009MHz. Z-stacks were captured with an interval size of 130.6\u2009nm and images were acquired at a resolution of 1,024\u2009\u00d7\u20091,024 pixels.<\/p>\n Image analysis<\/p>\n All images were processed as maximum intensity projections. Nuclei (DAPI) and paraspeckles (NEAT1_2) were segmented using CellProfiler v.4.2.6 with the Otsu thresholding method. The CV of the NEAT1_2 signal within nuclei and the number of segmented paraspeckles were used as independent metrics. Both metrics have their respective advantages and disadvantages. Although CV relies on more reliable nuclei segmentation, it is an indirect measure of paraspeckle integrity and does not provide precise structural information. In contrast, paraspeckle quantification directly assesses paraspeckle formation but is more error prone, particularly at later time points when the NEAT1_2 signal diminishes.<\/p>\n Line profile analysis and preparation of representative images were performed using Fiji (ImageJ v.2.16.0\/1.54p).<\/p>\n Subcellular localization analysis of viral protein interactors<\/p>\n To compare subcellular localizations of host interactors across studies, we compiled viral\u2013host protein interactions from SHVIP (this study), refs. 13<\/a>,15<\/a> and the meta-analysis in ref. 26<\/a>. For all datasets, only viral proteins that were identified in SHVIP (PB1, PB2, NP, NS1, M1, M2, HA and NA) were considered.<\/p>\n Host protein localizations were annotated based on data from the Human Protein Atlas. Only primary localization assignments were retained. A simplified set of localization categories was defined that grouped terms as follows: plasma membrane, cytosol, cytoskeleton, vesicular system, ER, mitochondrion, nucleoplasm, nucleoli, nucleus (other) and other, followed by manual curation. For ambiguous or multilocalized proteins, the most specific localization according to UniProt was assigned.<\/p>\n For SHVIP, we included only high-confidence host interactors filtered at 2% FDR at both the cross-linking PPI levels. For ref. 13<\/a>, host interactors were taken directly from their published AP\u2013MS dataset in HEK293 cells expressing individual FLAG-tagged viral proteins. For ref. 15<\/a>, we used the high-confidence filtered dataset provided by the authors, based on AP\u2013MS of 13 IAV proteins expressed in 3 cell types across 3 different IAV strains. For ref. 26<\/a>, only host interactors identified in at least three independent studies or databases were considered, as described in their meta-analysis.<\/p>\n Viral protein localization was manually curated from published literature (Supplementary References<\/a>) and database annotations (UniProt). Where conflicting evidence for localization existed, fractional assignments were split evenly across compartments (for example, a score of 1 distributed as 0.5 and 0.5 across 2 compartments).<\/p>\n Validation of cross-links by host structures and AlphaFold 2 models<\/p>\n For each cross-linked human protein, all available structures in the PDB were retrieved using the corresponding UniProt identifier via the UniProt83<\/a> REST API and RCSB PDB Search API84<\/a>. All cross-linked pairs were mapped to the retrieved structures, and if the corresponding pair was found among structures, C\u03b1\u2013C\u03b1 was calculated. If one pair was mapped to multiple structures, the shortest C\u03b1\u2013C\u03b1 distance was taken. The same mapping procedure was used for the random cross-linked pairs generated by taking arbitrary lysine\u2013lysine pairs for individual proteins and protein pairs detected in PPIs. AlphaFold 2 (ref. 44<\/a>) models were built using AlphaPulldown85<\/a>. Structural figures were rendered using UCSF ChimeraX86<\/a>.<\/p>\n Virus\u2013host interaction structural modelling<\/p>\n AlphaFold 2 models were built using AlphaPulldown85<\/a> v.2.0 using 24 recycles and 4 predictions per model. The sequence and template databases as of August 2024 were used. AlphaFold 3 was run using the original AlphaFold 3 code and using the default sequence database (downloaded in November 2024) and PDB database from November 2021 using the download script provided in AlphaFold 3 (ref. 31<\/a>). AF3x32<\/a>, a modelling protocol implemented within the AlphaFold 3 code that allows incorporation of cross-links as restraints in the form of explicit covalent ligands, was run with default settings and using version #dfb94a3 and the same databases as AlphaFold 3 (ref. 31<\/a>). For AlphaFold 3 and AF3x, 20 random seeds were used per prediction (in some cases up to 1,000). The quality of models was assessed using the interface-predicted template modelling (ipTM) score, predicted template modelling (pTM), residue\u2019s predicted local distance difference test (pLDDT) score and predicted alignment error, as returned by all the above tools. GRASP33<\/a> was run in normal mode using version 2a381bc, with four predictions per model, ranking by pTM. The multiple sequence alignment (MSA) and templates were the same as for AlphaFold2. Distance restraints in GRASP were applied using a 30\u2009\u00c5 threshold and 0.05\u2009FDR. Structural figures were rendered using UCSF ChimeraX86<\/a>.<\/p>\n Statistical analysis<\/p>\n Statistical analyses, plotting and graph preparation were performed using GraphPad Prism 10 (GraphPad Software). Exact P values for statistical analyses depicted in the figures are provided in the Source data<\/a>.<\/p>\n Other bioinformatics analyses<\/p>\n Functional enrichment analysis was performed using the Enrichr web platform87<\/a>. The default Fisher\u2019s exact test with the Benjamini\u2013Hochberg method for correction for multiple hypotheses testing was used. Overlap with prior host\u2013host interactions was calculated using BioGRID v.2.0.18 (ref. 88<\/a>). For cross-links to viral proteins where alternative proteins with identical cross-linked peptide sequences were detected ambiguously (for example, histone proteins), only the first protein from the list of ambiguous candidates was selected for the analysis. Cross-link diagrams were drawn using xiNET89<\/a>.<\/p>\n Reporting summary<\/p>\n