Shigella flexneri-Encoded E3 Ubiquitin Ligase IpaH2 Reveals Plakophilin-2 as a Host Restriction Factor for Sindbis Virus

Arthropod-borne viruses (arboviruses) cause a wide range of acute and chronic diseases and represent a growing global health burden. Although some vaccines exist, antiviral therapies remain limited. Identifying host restriction factors may enable new therapeutic strategies. We previously showed that bacterial effector proteins can serve as tools to uncover innate immune defenses. Here, we used a bacterial effector screen in bat cells to identify host factors restricting the arboviruses Sindbis virus (SINV) and O’nyong’nyong virus (ONNV). Several effectors enhanced infection by both viruses. However, we found the Shigella flexneri-encoded E3 ubiquitin ligase IpaH2 to selectively promote SINV replication. IpaH2 enhanced SINV infection across multiple mammalian cell lines, suggesting that it targets a conserved antiviral mechanism, and this effect required IpaH2 E3 ubiquitin ligase activity. Screening of putative IpaH2 host targets identified via ubiquitin-activated interaction trap (UBAIT) assays revealed the host factors ATP-binding cassette sub-family F member 3 (ABCF3) and Plakophilin-2 (PKP2) to play roles in restricting SINV infection. While ABCF3 broadly restricted multiple viruses, PKP2 specifically limited SINV replication, indicating a virus-specific restriction factor. These findings demonstrate that bacterial effector screening can identify both broadly acting and virus-specific host defenses, providing insight into antiviral mechanisms and potential therapeutic targets.