TRIM28 functions as SUMO ligase to SUMOylate TRAF6 and regulate NF-κB activation in HBV-replicating cells.

Background: Hepatitis B virus (HBV) is a pathogen that poses a serious threat to human health. The interaction between HBV and host has made great progress in recent years. SUMOylation is involved in virus-related cancer progression, but there are fewer studies on the mechanism of SUMOylation on HBV replication and antiviral defense. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a critical adaptor of the NF-κB pathways. Here, we focus on the roles of TRIM28 in regulating TRAF6 SUMOylation in HBV-replicating cells.

Methods: The SUMO1-modified TRAF6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated TRAF6 was detected by western blot using an anti-TRAF6 antibody. The interaction between TRAF6 and TRIM28 was identified by immunoprecipitation and LC-MS/MS. The modification sites of TRAF6 SUMOylation were identified by amino acid site mutation. Expression and localization of TRAF6 and TRIM28 were assessed by immunohistochemistry and immunofluorescence. The hydrodynamic injection HBV mouse model was used to determine the function of TRIM28-mediated TRAF6 SUMOylation in vivo.

Results: The results show that the levels of SUMO1-modified TRAF6 are elevated in HBV-replicating cells. Lys453 is a major SUMO1 modification site of TRAF6. There is an antagonistic interaction between SUMOylation and ubiquitination of TRAF6 protein. The SUMO ligase TRIM28 is responsible for catalyzing TRAF6 SUMOylation. Compared to the wild-type TRAF6, its SUMO site mutant TRAF6^K453R promotes NF-κB activation. Moreover, TRIM28 overexpression attenuates TRAF6-mediated NF-κB activation, thereby inhibiting HBV replication in vivo.

Conclusions: Our findings demonstrate that SUMO ligase TRIM28 affects the ability of TRAF6 on NF-κB activation, nucleocytoplasmic shuttling and HBV replication-related indicators. Our data reveal that TRIM28-mediated SUMOylation of TRAF6 is a novel mechanism to regulate the inflammatory response, which may pave the way for new strategies to control anti-HBV.