Fig 1: VPS34 knockdown inhibits coronavirus infection in vitro. (A–E) Multi-step growth curve of SARS-CoV-2. 293-ACE2 cells were transfected with a negative control shRNA or shRNAs targeting human VPS34 for 48 h and then infected with 0.02 MOI SARS-CoV-2 (WT, Beta, Delta, Omicron). Viral replication was monitored by: (A–D) RT-qPCR quantification of N gene copies in supernatants at indicated time. (E) Western blot analysis of SARS-CoV-2 N protein levels. (F) SARS-CoV-2 titer was determined by the TCID50 assay. (G–L) A549 cells were transfected with si NC, siVPS34, control vector (Ctrl), or myc-VPS34, and infected with 0.1 MOI HCoV-OC43 at indicated time points. (G, J) The HCoV-OC43 N protein expression was analyzed by Western blot using specific antibodies. (H, K) Viral load in supernatants was assessed through RT-qPCR targeting the HCoV-OC43 N gene. (I, L) Intracellular HCoV-OC43 N mRNA levels were quantified by RT-qPCR with GAPDH normalization. Data are presented as the mean ± SEM (n = 3). Statistical significance was analyzed using Student's t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Fig 2: VPS34-deficient mice are resistant to SARS-CoV-2 infection. (A–C) Vps34flox/+ and Vps34flox/+Sftpc cre mice were intranasally challenged with 2000 TCID50 mouse-adapted SARS-CoV-2. The survival (A) was monitored for 10 days (Vps34flox/+: n = 8, Vps34flox/+Sftpc cre: n = 9). The body weight (B) was monitored for 10 days (n = 6). (C–F) Lung tissues were collected at 3 dpi (n = 4). (C) SARS-CoV-2 N gene in lung tissues was determined by RT-qPCR. (D) Viral titer in lung tissues was determined by TCID50 assays. (E) Lung tissues fixed in 4% paraformaldehyde and H&E-stained at 3 dpi. (F) The expression of pro-inflammatory cytokines and chemokines was quantified by RT-qPCR. Data are presented as the mean ± SEM. Statistical analysis was determined by Student's t-test and the log-rank test for survival curves. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Fig 3: The discovery of potent and selective VPS34 inhibitors. (A) Schematic representation of group substitutions in SAR405. (B) Synthetic route for SAR405 derivatives. (C–E) Structural formulas of SAR405 derivatives and 3D views of their molecular docking with VPS34. (F–H) Interactions between SAR405 derivatives and VPS34 were analyzed using SPR.
Fig 4: Inhibition of VPS34 activity decreases coronavirus replication in vitro. (A, B) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with 0.02 MOI WT SARS-CoV-2 variant and simultaneously treated with VPS34 inhibitors (1 μmol/L). At 48 hpi, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained using an antiviral nucleocapsid (NP) antibody paired with an Alexa Fluor 488-conjugated secondary antibody (green). The nuclei were counterstained with DAPI (blue). Scale bars: 400 μm. (C–F) Dose-dependent inhibition of SARS-CoV-2 variants by VPS34 inhibitors. Vero E6 cells were infected with 0.02 MOI SARS-CoV-2 variants (WT, Beta, Delta, and Omicron) and simultaneously treated with serially diluted VPS34 inhibitors (VPS34-IN-I, VPS34-IN-2, PIK-III, SAR405). Viral RNA copies in supernatants collected at 48 hpi were quantified via RT-qPCR. Inhibition potencies (EC50) were calculated based on viral RNA reduction relative to DMSO controls. (G–J) Cytotoxicity evaluation of VPS34 inhibitors. Cell viability was assessed using the CCK-8 assay after 48-h compound exposure. Data represent mean ± SEM from three independent experiments (n = 3).
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