Fig 2: SGK1 inhibits PFV replication. (A to C) HT1080-Control and HT1080 stable expression cells (1 × 105) were infected with PFV (MOI = 0.6). At 48 h postinfection, 600 μL of the supernatants (A) or 1/10 infected HT1080 cell lines (B) were incubated with PFVL cells (1 × 105), the luciferase activity was measured 48 h later, and the rest of the infected cells were lysed for Western blotting (C). (D to F) HT1080-siControl and HT1080-siSGK1 cells (1 × 105) were infected with PFV (MOI = 0.6). At 24 h postinfection, 600 μL of the supernatants (D) or 1/10 infected HT1080 cells (E) were incubated with PFVL cells (1 × 105), and the luciferase activity was measured 48 h later. (F) The rest of the infected cells were lysed for Western blotting. (G to I) PFV (MOI = 0.6) infected Control and sg3 cell lines (1 × 105). At 48 h postinfection, 600 μL of the supernatants (G) or 1/10 infected HT1080 cells (H) were incubated with PFVL cells (1 × 105), and the luciferase activity was measured 48 h later. (I) The remaining infected cells were lysed for Western blotting. Data are expressed as the means ± standard deviations. Data are representative of three independent experiments. One-way ANOVA was used to perform the statistical test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3: Schematic diagram of SGK1 inhibiting PFV replication. PFV infection enhances the promoter activity of SGK1 through the Tas protein, and then upregulates the mRNA and protein levels of SGK1. On the one hand, SGK1 interacts with the Tas activation domain to affect its transcription activation function. On the other hand, the constitutively active form of SGK1 enhances the phosphorylation of PFV Gag which promotes Gag degradation via the lysosomal pathway.

Fig 4: SGK1 inhibits the function of Tas activation domain. (A) HEK293T cells (5 × 105) were transfected with Flag-Tas (1 μg) and empty vector or HA-SGK1 (1 μg). At 48 h posttransfection, cells were incubated with or without the cross-linking chemical. Cell lysates were analyzed by Western blotting. The positions of Flag-Tas monomers (M) and dimmer (D) are indicated. (B) HEK293T cells (4 × 106) were transfected with Myc-Tas (3 μg), Flag-Tas (3 μg), and empty vector or HA-SGK1 (3 μg). At 48 h posttransfection, co-immunoprecipitation was performed using Flag antibodies. Western blotting of samples from cell lysates and immunoprecipitates using Myc, Flag, and HA antibodies. (C) PFVL cells (9 × 105) were co-transfected with HA-SGK1 (5 μg), Flag-Tas (5 μg), Flag-Tas (5 μg) + HA-SGK1 (5 μg), respectively. At 48 h posttransfection, cells were subjected to a ChIP assay as described in Materials and Methods. Subsequently, PCR amplification was carried out to detect LTR in the immunoprecipitated chromatin fragments. (D) Part of the cell lysates was used to detect protein levels using Western blotting. (E) HEK293T cells (2.5 × 106) were co-transfected with PFV IP-Luc (3 μg) and HA-SGK1 (5 μg), Flag-Tas (5 μg), Flag-Tas (5 μg) + HA-SGK1 (5 μg), respectively. At 48 h posttransfection, cells were subjected to a ChIP assay. Subsequently, PCR amplification assay was carried out to detect IP in the immunoprecipitated chromatin fragments. (F) Part of the cell lysates was used to detect protein levels using Western blotting. (G) HEK293T cells (2 × 105) were transfected with pCMV-BD or pCMV-BD-Tas (223 to 300 aa) (0.02 μg), HA-SGK1 or empty vector (0.3 μg), pFR-Luc (0.01 μg) and pCMV-β-gal (0.025 μg). After 48 h of transfection, luciferase activity was measured. (H, I) HEK293T cells (2 × 105) were transfected with pCMV-AD or pCMV-AD-Tas (1 to 220 aa) (0.3 μg), HA-SGK1 or empty vector (0.3 μg), LTR-Luc (0.05 μg) (H) or IP-Luc (0.025 μg) (I), and pCMV-β-gal (0.025 μg). After 48 h of transfection, luciferase activity was measured. Data are expressed as the means ± standard deviations. Data are representative of three independent experiments. One-way ANOVA was used to perform the statistical test. ****, P < 0.0001; ns for P > 0.05.

Fig 5: Non-human SGK1 inhibits PFV replication. (A) Homology of human and bovine SGK1 proteins. Asterisks (*) denote identical amino acids. (B) HEK293T cells (2 × 105) were co-transfected with pcPFV (1.5 μg) and empty vector, hSGK1 or bSGK1 (0.5 μg). At 48 h posttransfection, 600 μL of the supernatants or 1/20 transfected cells were incubated with PFVL cells (1 × 105), the luciferase activity was measured 48 h later, and the rest of transfected cells were lysed for Western blotting. (C, D) HEK293T cells (2 × 105) were co-transfected with BFV (1.5 μg) and empty vector, hSGK1 or bSGK1 (0.5 μg). At 48 h posttransfection, 1/20 transfected cells were incubated with BFVL cells (1 × 105), the luciferase activity was measured 48 h later (C). (D) The remaining transfected cells were lysed for Western blotting. Data are expressed as the means ± standard deviations. Data are representative of two independent experiments. One-way ANOVA was used to perform the statistical test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.