Fig. 4: The antiviral capacity of human and bat GBP1.

A Surface potential predictions for GBP1 structures were predicted using AlphaFold and made using the APBS server. Positive charge is indicated in blue, negative charge is in red. Arrows indicate the region of differential charge between GBP1 proteins. B HEK293T cells were transfected with 250 to 1000 ng of plasmid encoding for either P. alecto (Pa), E. fuscus (Ef), or human (Hu) GBP1 tagged with 3xFLAG for 48 h. Cell lysates were then probed for FLAG-GBP1 and ACTB. C HEK293T cells were transfected with 500 ng of plasmid encoding PaGBP1, EfGBP1, or HuGBP1 for 48 h, followed by methanol fixation. Cells were stained for FLAG-GBP1, ACTB, and DAPI and visualized by confocal microscopy. Scale bars = 25 µm. D A549-ACE2 cells were transfected for 24 h with 250 to 1000 ng of HuGBP1, PaGBP1, or EfGBP, followed by infection with the indicated viruses. Infection intensity was assessed by measuring GFP signal (VSV-GFP), TCID₅₀ assay (HSV-1, SARS-CoV-2, MERS-CoV), or by plaque assay (VACV, EfPV). Mean values are indicated within the boxes. E Schematic representation of wild-type GBP1 and the A₁₈AA substitution mutant. Surface charge potential for mutated GBP1 proteins predicted using AlphaFold and the APBS server are shown. Positive charge is indicated in blue, and negative charge is shown in red. The site of mutagenesis is contoured in black on the protein surface. Image created in BioRender [https://BioRender.com/o9oes1x]. F A549-ACE2 cells were transfected with 250 to 1000 ng of WT PaGBP1 or EfGBP1 and the respective A₁₈AA mutant for 24 h, followed by infection with EfPV (MOI 0.01) for 48 h. Supernatant was titered by plaque assay (One-way ANOVA, Tukey’s range test, ****<0.0001). Data were represented as mean ± SD, n = 3 biological replicates. G Representative plaques for PaGBP1 and EfGBP1 from undiluted supernatant.