Figure 1.

ASFV protein I215L inhibits IFN-I signaling pathway. (A) I215L transcripts were detected in mock-infected or infected PAM cells by qRT-PCR. PAMs were mock-infected or infected with the virulent Arm/07/CBM/c2 ASFV strain (2 PFU/cell) and collected at 2, 4, 8, or 16 h post-infection for mRNA extraction. As a control of an early-late and late viral gene expression, CP204L and B646L mRNA were measured, respectively. (B) I215L gene encodes for an early protein detected from 4 hpi by Western blot. Arm/07/CBM/c2 infected PAMs (2 PFU/cell) were collected at 4, 8, or 16 hpi, lysed in RIPA buffer and analyzed by Western blot. Antibodies against ASFV-pI215L, p72 (ASFV late protein) and actin were used. pI215L levels were quantified according with their actin levels and relativized to the 4 hpi sample by using ImageJ. (C) ISRE-luciferase reporter experiment was performed in HEK-293 cells transfected with empty vector control (EV) or with increasing concentrations of I215L (0.3, 1, or 3 μg/1 × 10⁶ cells) in absence or in presence of IFN-I (500 U/mL). Graph represents the means of the Firefly luciferase RLU (Relative Luminiscence Units) values divided by its Renilla luciferase RLU values from biological triplicates. Obtained values were relativized against the untreated EV transfected sample. (D) ISG15 mRNA levels were analyzed in COS-1 cells transfected with EV or increasing concentrations of pI215L (0.5 or 2.5 μg/1 × 10⁶ cells) in absence or in presence of IFN-I by qRT-PCR. (E) I215L mRNA levels were amplified by qRT-PCR in the same conditions than (D) to verify the efficient I215L transcription in transfected HEK-293 cells. All data reflect mean ± SEM (n = 3). Data were statistically analyzed by using a Student t-test (**p < 0.01; ***p < 0.001).