The IFITM family of proteins undergoes various post-translational modifications, including palmitoylation, ubiquitination, phosphorylation, and methylation. These modifications are highly conserved across species and play critical roles in the functions of IFITMs by regulating intracellular localization, oligomerization, and biological activity. Palmitoylation modifications have been shown to impact the antiviral activity of IFITM1 derived from both porcine and murine sources.
To further investigate the conservation of IFITM1 across species, we conducted a comparative analysis of human, porcine, and murine IFITM1 proteins. Our findings reveal significant conservation at the sites of phosphorylation and palmitoylation modifications. To determine the impact of various modification sites on the antiviral activity of IFITM1, we generated four mutant plasmids of human IFITM1 (S16E，C2A，C3A) (Fig. H,I). Immunofluorescence assay showed that both wild-type and S16E variants of IFITM1 overexpression accurately localized around the cellular membrane while mutations at C2A and C3A sites resulted in improper localization to the cell membrane.
Finally, we investigated whether palmitoylation affects JEV replication by infecting HEK293T cells overexpressing wild-type or mutation IFITM proteins. The results demonstrate that palmitoylation site mutations impair the antiviral function of IFITM1.
