Mapping of molecular interactions between human E3 ligase TRIM69 and Dengue virus NS3 protease using hydrogen–deuterium exchange mass spectrometry

Tripartite motif (TRIM) E3 ligases target specific substrates, including viral proteins, for proteasomal degradation, and are thus essential regulators of the innate antiviral response. TRIM69 ubiquitinates the non-structural NS3 protein of Dengue virus for its degradation by the host machinery. This antiviral strategy abrogates the immunosuppression mediated by the NS2B–NS3 protease complex. To understand how this host-driven antiviral response against Dengue virus, we sought to define the mode of interaction between human TRIM69 and Dengue NS2B–NS3 and the subsequent polyubiquitination of the protease by the E3 ligase. We show that NS2B–NS3Δpro is sufficient as a substrate for ubiquitination by TRIM69 using ELISA and in vitro assays. Using hydrogen–deuterium exchange mass spectrometry (HDXMS), we mapped the interface of the interaction between TRIM69 and NS2B–NS3Δpro, and propose a rationale for the binding and subsequent ubiquitination process. Furthermore, through sequence analysis, we showed that the regions targeted by TRIM69 on the DENV-2 NS3 protease (NS3Δpro) are well conserved across DENV serotypes and other flaviviruses, including Zika virus, West Nile virus, and Japanese encephalitis virus. Our results show the direct interactions of TRIM69 with viral proteins, provide mechanistic insights at a molecular level, and highlight the functional relevance of TRIM69 interacting with the Dengue viral protein. Collectively, our findings suggest that TRIM69 may act as a pan-antiflaviviral restriction factor.