Genome-wide CRISPR screen identifies RNF24 as a critical host factor for foot-and-mouth disease virus entry

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  FIGURE 1. Genome-wide CRISPR screen identifies host factors required for FMDV infection. (A) Schematic of the porcine genome-wide CRISPR/Cas9 knockout screen in LLC-PK1-Cas9 cells. Cells were subjected to three rounds of FMDV challenge at increasing MOIs (0.00001, 0.001, 0.01). Surviving cells were collected for sgRNA amplification and sequencing. (B) Venn diagram showing the overlap of the top 50 enriched sgRNAs identified across three independent screening experiments. (C) Viral RNA levels (qPCR) in pooled mutant cell lines targeting 17 candidate genes following FMDV infection (MOI = 0.01, 12 h), normalized to non-targeting sgRNA controls. (D,E) VP1 protein expression (Western blot) in WT and candidate gene-knockout LLC-PK1 cells at 12 h post-infection (MOI = 0.1). Tubulin or actin was used as a loading control. (F) Genotyping of homozygous RNF24-KO cells by Sanger sequencing. (G) Time-course analysis of VP1 expression (Western blot) in RNF24-KO and WT cells at 4, 8, and 12 h post-infection (MOI = 0.1). (H,I) Viral infectivity (plaque assay) in culture supernatants of RNF24-KO and WT cells at 8 h and 12 h post-infection (MOI = 0.1). (J) FMDV 3D copy number (qPCR) in RNF24-KO versus WT…

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  FIGURE 2. RNF24 knockout impairs FMDV adsorption and internalization. (A) FMDV RNA kinetics (qPCR) in RNF24-KO and WT cells from 0 to 4 h post-infection (MOI = 0.1). (B) Viral attachment assay. RNF24-KO and WT Cells were incubated with FMDV at 4 °C for 1 h (MOI = 10), washed, and analyzed for bound viral RNA by qPCR. (C) Internalization assay. WT and RNF24 KO cells were adsorbed with FMDV (MOI = 10) at 4°C for 1 h. After shifting to 37°C for 1 h to allow entry, cells were subjected to three washes with an ice-cold alkaline high-salt solution (1 M NaCl, 50 mM sodium bicarbonate, pH 9.5) to strip residual surface-bound virions, followed by three final washes with PBS. Internalized viral RNA was measured by qPCR. (D) Confocal microscopy detecting dsRNA in RNF24-KO and WT cells at 3 h post-infection (MOI = 10). Scale bars: 20 µm. (E,F) Intracellular and extracellular viral titers (plaque assay) in RNF24-KO and WT cells at 12 h post-infection (MOI = 0.1). All experiments were independently repeated at least three times (n = 3), with representative images/blots shown. Quantitative data are expressed as mean ± SD. Statistical significance was determined by Student’s t test, and specific p-values…

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  FIGURE 3. RNF24 modulates integrin-mediated signaling to regulate FMDV entry. (A) Transcript levels of HSPGs and integrins in RNF24-KO versus WT cells (qPCR). (B,C) Protein levels of ITGB1 (B) and ITGAV (C) in RNF24-KO and WT cells, with or without FMDV infection (Western blot). (D) Transcript levels of HSPGs and integrins in RNF24-overexpressing (OE) versus control LLC-PK1 cells (qPCR). (E) Protein levels of ITGAV and ITGB1 in RNF24-OE and control LLC-PK1 cells, with or without FMDV infection (Western blot). (F-H) Co-IP analysis in HEK293T cells co-transfected with RNF24-Flag and HA-tagged ITGAV or ITGB1, or corresponding empty vectors for 24 h. (I) Confocal microscopy showing colocalization of RNF24-Myc (red) with HA-ITGAV or HA-ITGB1 (green) in HEK293T cells. Scale bars: 2 µm or 5 µm. (J) Co-IP analysis in HEK293T cells co-transfected with GST-VP1 and RNF24-Flag or empty vector for 24 h. (K,L) Co-IP analysis in HEK293T cells co-transfected with HA-ITGAV (K) or HA-ITGB1 (L), GST-VP1, and RNF24-Flag or empty vector for 24 h. All experiments were independently repeated at least three times (n = 3), with representative images/blots shown. Quantitative data are expressed as mean ± SD.…

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  FIGURE 4. RNF24 interacts with LPXN. (A) Potential ubiquitination substrates of RNF24 predicted by UbiBrowser 2.0. (B,C) Co-IP analysis in HEK293T cells co-transfected with HA-LPXN and RNF24-Flag or empty vector for 24 h. (D) Confocal microscopy showing colocalization of RNF24-Myc (red) with HA-LPXN (green) in HEK293T cells. Scale bar: 5 µm. (E) Quantification of fluorescence intensity at RNF24 and LPXN colocalized sites. (F) LPXN expression (Western blot) in RNF24-overexpressing LLC-PK1 cells with or without FMDV infection. (G) LPXN expression (Western blot) in RNF24-KO and WT cells. (H) LPXN protein stability assay. HA-LPXN and RNF24-Flag were co-expressed in HEK293T cells, followed by cycloheximide (CHX) chase treatment for the indicated durations and Western blot analysis. All experiments were independently repeated at least three times (n = 3), with representative images/blots shown.

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  FIGURE 5. RNF24 preferentially promotes K27-linked ubiquitination of LPXN at residue K162. (A) Co-IP analysis in HEK293T cells co-transfected with LPXN-Flag and HA-ubiquitin (HA-Ub; WT or indicated mutants) or empty vector for 24 h. (B) Immunoblot analysis of lysates from HEK293T cells expressing LPXN-Flag and HA-Ub (WT or mutants) for 24 h. (C) Co-IP analysis in HEK293T cells co-expressing HA-Ub, LPXN-Flag, and Myc-RNF24 or empty vector for 24 h. (D) Co-IP analysis in RNF24-KO versus WT cells co-transfected with HA-Ub, Flag-LPXN, and Myc-RNF24 or empty vector for 24 h. (E) Co-IP analysis in HEK293T cells expressing HA-Ub (WT or mutants), Flag-LPXN, and Myc-RNF24 or empty vector for 24 h. (F) Co-IP analysis in HEK293T cells expressing Myc-Ub (WT or mutants), HA-LPXN, and RNF24-Flag or empty vector for 24 h. (G) Schematic diagram of LPXN ubiquitination sites and point mutations. (H) Co-IP analysis in HEK293T cells expressing HA-Ub, LPXN-Flag (WT or mutants), and RNF24-Myc or empty vector for 24 h. (I) Subcellular fractionation and Western blot analysis of HA-LPXN in cytosolic and membrane fractions from HEK293T cells expressing HA-LPXN with or without RNF24-Flag for 24 h. (J) Subcellular…

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  FIGURE 6. LPXN is required for FMDV entry. (A) VP1 expression (Western blot) in LPXN-overexpressing (OE) and control SK6 cells at 12 h post-infection (MOI = 0.1). (B) Viral infectivity (plaque assay) in supernatants of LPXN-OE and control SK6 cells at 12 h post-infection (MOI = 0.1). (C) LPXN protein levels (Western blot) in SK6 cells at 48 h after transduction with shRNA lentiviruses targeting LPXN. (D) VP1 expression (Western blot) in shLPXN and shNC transduced SK6 cells at 12 h post-infection (MOI = 0.1). (E) Viral infectivity (plaque assay) in supernatants of shLPXN and shNC transduced SK6 cells at 12 h post-infection (MOI = 0.1). (F) Viral attachment assay. shLPXN and shNC transduced SK6 cells were incubated with FMDV (MOI = 10) at 4 °C for 1 h, washed, and analyzed for bound viral RNA by qPCR. (G) Internalization assay. shLPXN and shNC transduced SK6 cells were adsorbed with FMDV (4 °C, 1 h, MOI = 10), After shifting to 37°C for 1 h to allow entry, cells were subjected to three washes with an ice-cold alkaline high-salt solution (1 M NaCl, 50 mM sodium bicarbonate, pH 9.5) to strip residual surface-bound virions, followed by three final washes with PBS. Internalized viral RNA was…

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  FIGURE 7. Ternary interaction among LPXN, VP1, and ITGAV/ITGB1. (A,B) Co-IP analysis in HEK293T cells co-transfected with LPXN-Flag and HA-VP1 for 24 h. (C) Confocal microscopy showing colocalization of LPXN-Flag (red) with HA-VP1 (green) in HEK293T cells. Scale bar: 5 µm. (D) Fluorescence intensity quantification at LPXN-VP1 colocalized sites. (E,F) Co-IP analysis in HEK293T cells expressing GST-VP1, HA-ITGAV (E), or HA-ITGB1 (F), with LPXN-Flag or empty vector for 24 h. (G) Confocal microscopy visualizing pairwise colocalization among HA-ITGAV (red)/HA-ITGB1 (red), VP1 (GFP-green), and LPXN-Flag (magenta) in HEK293T cells. Scale bars: 2 µm or 5 µm. (H) Fluorescence intensity quantification at sites of triple colocalization (LPXN, VP1, and ITGAV/ITGB1). (I) Schematic diagram of VP1 truncation mutants. (J) Co-IP analysis in HEK293T cells expressing LPXN-Flag with GST-VP1 (WT or truncated forms) or empty vector for 24 h. (K, L) Co-IP analysis in HEK293T cells expressing HA-ITGAV (K) or HA-ITGB1 (L) with GST-VP1 (WT or truncated forms) or empty vector for 24 h. All experiments were independently repeated at least three times (n = 3), with representative images/blots shown.

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  FIGURE 8. Lysine 162 of LPXN is critical for VP1-integrin interaction. (A) Co-IP analysis in shLPXN and shNC SK6 cells co-transfected with GST-VP1, HA-ITGAV/HA-ITGB1, or empty vector for 24 h. (B) Confocal microscopy showing colocalization of GST-VP1 (green) with HA-ITGAV or HA-ITGB1 (red) in shNC, shLPXN, and LPXN-rescued (LPXN-R) SK6 cells co-transfected with the indicated constructs for 24 h. Scale bar: 5 µm. (C) Co-IP analysis in HEK293T cells expressing GST-VP1, HA-ITGAV/HA-ITGB1, and Flag-LPXN (WT or K162R mutant) or empty vector for 24 h. (D) Proposed cellular model for RNF24 in FMDV entry. Left: RNF24 promotes K27-linked polyubiquitination of LPXN at K162, promoting its membrane translocation and scaffolding of an integrin-LPXN-VP1 ternary complex, which stabilizes virus-receptor binding and facilitates internalization. Right: Loss of RNF24 reduces membrane-localized LPXN, disrupts ternary complex assembly, and inhibits viral entry. All experiments were independently repeated at least three times (n = 3), with representative images/blots shown.