Contribution to the study of the role of the E3 Ubiquitin Ligase TRIM28 (KAP1) in the Innate Immune Response Against RNA Viruses

Tripartite motif-containing protein 28 (TRIM28), also known as Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is a multifunctional regulator that integrates epigenetic silencing with post-translational modifications (PTMs), including SUMOylation and ubiquitination. Initially identified as a transcriptional corepressor that maintains heterochromatin through SETDB1- and HP1-associated complexes, TRIM28/KAP1 has more recently been implicated in viral infection and host innate immunity. Accumulating evidence indicates that TRIM28/KAP1 exerts context-dependent effects during viral infection, functioning either as a restriction factor or as a facilitator of viral replication depending on the cellular and viral context. The RIG-I-like receptor (RLR) signaling pathway, a central axis of antiviral innate immunity against RNA viruses, relies on mitochondrial antiviral signaling protein (MAVS) to transmit signals leading to interferon production and is tightly regulated by ubiquitination. However, the specific target of TRIM28 within the RLR signaling pathway and its functional relevance during RNA virus infection in vivo remain unclear. Therefore, this thesis aims to elucidate the role of TRIM28 in RNA virus-induced antiviral innate immunity, with a focus on its regulation of the RIG-I–MAVS axis.
In the first part of this study, we characterized the functional role of TRIM28 in RNA virus-induced antiviral responses. TRIM28 expression was dynamically upregulated following RNA virus infection. Functional analyses showed that TRIM28 acts as a negative regulator of antiviral immunity: its overexpression suppressed type I interferon production and interferon-stimulated gene expression, whereas its knockdown enhanced these responses. Consistent with this role, TRIM28 promoted the replication of RNA viruses, including vesicular stomatitis virus (VSV) and enterovirus 71 (EV-71). In vivo, lung-specific knockdown of TRIM28 further demonstrated that TRIM28 deficiency enhanced antiviral responses, reduced viral burden, and alleviated virus-induced pulmonary pathology, supporting its role in promoting viral pathogenicity.
At the mechanistic level, we investigated how TRIM28 modulates antiviral signaling pathways. TRIM28 suppressed TBK1 and IRF3 activation, leading to reduced IRF3 nuclear translocation and attenuation of downstream interferon responses. Further analyses identified MAVS as the key signaling node targeted by TRIM28, and TRIM28 specifically interacted with MAVS. RNA virus infection induced CRM1-dependent nuclear export of TRIM28, allowing its accumulation in the cytoplasm and facilitating its interaction with MAVS. TRIM28 then promoted RBCC domain-dependent K48-linked polyubiquitination of MAVS, leading to its proteasomal degradation and suppression of downstream TBK1-IRF3 signaling. Mutational analyses further identified Lys136 and Lys461 of MAVS as critical sites required for TRIM28-mediated ubiquitination.
In summary, this study identifies TRIM28 as a negative regulator of type I interferon responses during RNA virus infection. Mechanistically, TRIM28 undergoes CRM1-dependent nuclear export and targets MAVS for RBCC domain-dependent K48-linked ubiquitination and degradation, thereby attenuating downstream antiviral signaling. Lys136 and Lys461 on MAVS are further identified as key sites required for this regulation. These findings establish MAVS as a direct substrate of TRIM28 and provide new insight into how ubiquitin-dependent mechanisms fine-tune antiviral innate immunity.