Newcastle disease virus acquires phosphatidylserine through the budding process to enhance infectivity

Figure 1.. Plasma-membrane PS is critical for NDV budding from the host cell (A) A549 cells seeded in 96-well plates were treated with the indicated concentrations of Fendiline for 24 h, and viability was measured with the CCK-8 assay (n = 3 wells per group). (B) After 48 h of Fendiline treatment, cells were permeabilized, labelled with Annexin V – fluorochrome and total ps was quantified by flow cytometry. (C) HEK-293T cells were co-transfected with plasmids encoding M and the PS probe Lact-C2-GFP. At 24 h post-transfection (hpt) Fendiline was added; colocalization of M (red) and PS (green) was assessed by confocal microscopy. Scale bar, 10 µm. (D) HEK-293T cells transfected with M alone were treated with DMSO or Fendiline (1 or 5 µM) for 48 h. VLPs and cell lysates were harvested and analysed by Western blot. The budding index (bottom) was calculated as M in VLPs/(M in VLPs + M in lysate) normalized to DMSO. (E and F) A549 cells were pre-treated with Fendiline or vehicle, infected with NDV (MOI = 0.1) and analysed 24 hpi by Western blot (E) and RT-qPCR for NP mRNA(F). Data are presented as mean ± sem of three independent experiments. Statistical significance was determined by one-way…

Figure 2.. PS is a key phospholipid in NDV infection. (A–C) A549 cells transfected with CRISPR/Cas9 knockout plasmids targeting PSS1 or PSS2. were infected with NDV (MOI = 0.1). NP mRNA and protein were measured at 12 hpi by RT-qPCR (A) and Western blot (B); viral titres were determined by TCID₅₀ (C). (D–F) Over-expression of PSS1/2 was achieved by plasmid transfection (24 h) before infection (MOI = 0.1); NP mRNA (D), protein (E) and titres (F) were analysed as above. (G and H) Cells were pre-incubated with the indicated concentrations of exogenous PS (20 μM) for 2 h, infected with NDV (MOI = 0.1) and maintained in PS-containing medium. NP protein and infectious titer were assessed at 12 hpi by western blot (G) and plaque assay (H).

Figure 3.. Surface-exposed PS on NDV mimics an “eat-me” signal. (A) Virions purified from 24 h culture supernatants by sucrose-gradient centrifugation were subjected to LC-MS/MS for PS detection. (B) Negative-stain TEM shows intact NDV (left) and virions pre-blocked with Annexin V (right). (C) dot-blot of serially diluted purified virions probed with anti-PS or anti-NP antibody. (D) A549 cells were incubated with NDV (50 MOI) pre-treated with Annexin V (20 µg); adsorbed genomes were quantified by RT-qPCR. (E) Dose-dependent inhibition of infection (0.1 MOI) by Annexin V (20 µg) was assessed by Western blot for NP (12 hpi). (F) Virions were pre-incubated with Annexin V (20 µg) before infection (0.1 MOI); NP mRNA was measured at 12 hpi. (G) Virions were pre-incubated with increasing concentrations of anti-PS antibody for 2 h before infection (0.1 MOI); NP protein was analysed by Western blot.

Figure 4.. NDV infection drives TMEM16F-dependent PS externalization. (A–D) A549 cells were infected with NDV or left uninfected. Surface PS was quantified at the indicated time points by flow cytometry (A, B) and fluorescence microscopy (C, D). In A and C, infections were performed at an MOI of 1 and sampled at 12 and 24 hpi; in C and D, infections were performed at an MOI of 0.01, 0.1, 1, 3, and 5 and sampled at 12 hpi. (E and F) Cytosolic Ca2 + was monitored with Fluo-4 AM under identical conditions.

Figure 5.. Ca2+-activated TMEM16F is implicated in the NDV-induced externalization of PS. (A) CCK-8 assays on A549 cells were performed to evaluate the BAPTA-AM toxicity. (B) Cells were either pre-treated with BAPTA-AM (4 μM) or mock-treated, following infection of NDV (1 MOI). Extracellular PS levels were analyzed by FCM (12 hpi). (C and D) Efficiency of siRNA interference with TMEM16F expression was evaluated by RT-qPCR and western blot. (E) Extracellular PS levels after interference with TMEM16F expression levels. Data are presented as mean ± sem (n = 3). Significance was analyzed by unpaired Student’s t-test (p < 0.01). (F) CCK-8 assays on A549 cells were performed to evaluate Ionomycin toxicity. (G) A549 cells were transfected with a plasmid encoding TMEM16F for 24 h, then treated with Ionomycin (4 μM) or left untreated. Extracellular PS levels were then measured using FCM. (H) CCK-8 assays on A549 cells were performed to evaluate Niclosamide toxicity. (I) A549 cells were transfected with a plasmid encoding TMEM16F for 24 h, followed by infection with NDV (1 MOI). Subsequently, the cells were treated with either BAPTA-AM or Niclosamide(4 μM) or left untreated. Extracellular PS levels…

Figure 6.. The HN protein is essential for NDV-triggered PS externalization. (A) Expression of NDV structural and non-structural proteins were analysed by Western blot. (B) A549 cells transfected with plasmids encoding individual NDV proteins for 36 h were analysed for surface PS by flow cytometry. (C) Cells transfected with HN, infected with NDV, or treated with endoplasmic reticulum stress inducer thapsigargin (4 µM) were loaded with Fluo-4 am and imaged. (D) Co-immunoprecipitation of HA-TMEM16F and Flag-HN after co-transfection in HEK-293T cells. (E and F) Cells expressing HN, F or M were loaded with Fluo-4 AM and analyzed by fluorescence microscopy (E) and flow cytometry (F).

Figure 7.. PS externalization enhances NDV replication. (A-C) A549 cells over-expressing TMEM16F were infected with NDV (MOI = 0.1); NP mRNA, protein and viral titres were determined at 12 and 24 hpi. (D–F). TMEM16F was silenced with siRNA prior to infection and analysed as above. (G and H). Cells were pre-treated with Niclosamide (4 µM) or vehicle before infection; replication was monitored by immunofluorescence (G) and western blot (H). (I) Virions released from Niclosamide- or mock-treated cells (24 hpi) were concentrated, NP expression levels equalised, and PS content determined by dot-blot with anti-PS antibodies. (J) Virions released from Niclosamide- or mock-treated cells (24 hpi) were collected and viral genomes were quantified by qRT-PCR. After normalizing to equal genome copies, specific infectivity (PFU/genome copy) was determined by plaque assay.

Figure 8.. TYRO3 and TIM-4 function as PS receptors to mediate NDV entry into A549 cells (A) A549 cells transfected with the indicated siRnas were infected with NDV (MOI = 50). NP mRNA was quantified at 1 hpi to assess entry. (B) Over-expression of TYRO3/GAS6, TYRO3/PROS1 or TIM-4 were infected with NDV (MOI = 50). Data are presented as mean ± SEM of three independent experiments (n = 3). Statistical significance was determined by one-way anova test; p < 0.05, p < 0.01 vs. vector control group. NP mRNA was quantified at 1 hpi to assess entry. (C and D) fluorescence intensity of RFP-NDV (C) or GFP-NDV (D) was quantified after silencing or over-expressing TYRO3/TIM-4. (E–H) NP protein levels were measured by western blot under identical conditions. (I) A549 cells were pre-incubated with increasing concentrations of anti-TIM-4 antibody for 1 h before infection; NP protein was analyzed at 12 hpi.