Antiretroviral mechanisms of TRIM56 - PROJECT SUMMARY/ABSTRACT The innate immune effector TRIM56 restricts a broad range of viruses, yet its molecular mechanisms remain elusive. I propose to uncover the underlying mechanism of TRIM56’s antiviral activity, with a focus on HIV-1, where its role is better understood. TRIM56 is an E3 ubiquitin ligase, which typically mediates the transfer of ubiquitin to a target substrate with potential impacts on stability, binding, or trafficking of the target protein. Unlike most E3 ligases, which utilize protein-interaction domains for substrate recognition, TRIM56 utilizes an NHL domain known for its RNA binding activity. This unique feature suggests that TRIM56 may employ an unconventional mode of target recognition, possibly involving direct interaction with viral RNA. My preliminary data reveal that TRIM56 interacts with the HIV-1 genome similarly to the viral packaging protein Gag, and that the ubiquitin ligase function of TRIM56 is important for restricting HIV-1. While these data do not directly demonstrate whether TRIM56 NHL binds RNA directly or indirectly, they support the two following hypotheses: (a) TRIM56 may directly bind the HIV-1 RNA, thereby blocking Gag binding and subsequent viral packaging, while ubiquitinating nearby proteins co-occupying the RNA to limit viral RNA stability or translation; (b) TRIM56 may bind HIV-1 RNA indirectly through Gag, and subsequently ubiquitinate Gag to promote its degradation. To distinguish between these hypotheses, I will (I) investigate the mode of viral genomic RNA recognition (whether direct or indirect) using biochemistry and structural biology; (II) determine the ubiquitination target of TRIM56 using proteomic analyses and viral translation and packaging assays. The proposed research promises to identify a novel mechanism for viral RNA detection and restriction. It will also lay the groundwork for further exploration of TRIM56’s actions against other viruses, with the ultimate goal to identify a unifying mechanism for broad viral sensing. I will carry out this research in the laboratory of Dr. Sun Hur at Boston Children’s Hospital (BCH)/Harvard Medical School (HMS). Dr. Hur is an expert on innate immune response to pathogen nucleic acids, and her lab additionally hosts expertise in protein biochemistry and structural biology. The BCH/HMS environment supports postdoctoral research fellows with numerous core facilities, technique trainings, and career development workshops, in addition to supporting a rich intellectual environment in which many labs collaborate. I will leverage the surrounding expertise, research seminars, and professional development courses during the fellowship period to strengthen my research and to prepare to independently head a lab.
