# Investigating MHC-I downregulation by HIV-1 Nef using cryo-electron tomography with laser-based phase contrast > **NIH NIH F32** · UNIVERSITY OF CALIFORNIA BERKELEY · 2023 · $69,500 ## Abstract Project Summary/Abstract Nef is a viral accessory protein which plays a critical role in the pathogenesis of HIV. Among other functions, it undermines the host immune response by downregulating MHC-I expression at the cell surface. By binding the clathrin adaptor AP-1 and its cofactor, the GTPase Arf1, Nef drives the formation of AP-1:MHC-I complexes. This sequesters MHC-I in clathrin-coated vesicles which are eventually trafficked to the lysosome. However, this mechanism is only partially understood. Cryo-electron tomography (cryo-ET) is a powerful method which can visualize the interactions between AP-1 and Nef on physiological membranes. Recent work in the Hurley lab used cryo-ET to visualize the interactions between Nef and AP-1 on membranes in vitro, finding that the complex forms a lattice. The reconstruction indicated that Nef stabilizes, but is not essential for, lattice formation, suggesting that an AP-1:Arf1 lattice forms as part of normal clathrin-mediated trafficking and that this lattice is hijacked by Nef to downregulate MHC-I. However, the resolution remains limited by the poor signal-to-noise ratio (SNR) of cryo-ET. In the proposed work, the SNR of cryo-ET will be increased in order to test this hypothesis with unprecedented resolution. To increase SNR, the laser phase plate (LPP), a technology developed in the Müller lab, will be integrated into a latest-generation aberration-corrected cryo-electron microscope. In Aim 1, the LPP will be benchmarked against standard, defocus-based cryo-electron microscopy by performing single- particle reconstructions of the purified model protein, apoferritin. This will demonstrate the first atomic resolution imaging with the LPP and thus introduce a powerful new imaging modality to the structural biology toolbox. In Aim 2, the work of the Hurley lab will be built upon by using the new imaging method of laser-based phase contrast cryo-ET to solve the structures of membrane-bound AP-1:Arf1 complexes in both the presence and absence of Nef. Comparison of the observed structures will elucidate the mechanism by which AP-1 assemblies are co-opted by Nef to downregulate MHC-I. In addition, these reconstructions will provide the first structural data of AP-1:Arf1 complexes on membranes and may reveal the structural basis for AP-1 and Arf1 residues associated with other human disease. More broadly, the proposed research will introduce a new technology, laser-based phase contrast cryo-ET, to the structural biology community. This work will also provide deep structural insights into trafficking in healthy and HIV-infected cells, which may be instrumental in uncovering the mechanisms of HIV pathogenesis and inform therapeutic approaches. This research will leverage the expertise of the Hurley lab in membrane reconstitution and HIV biology as well as the unique electron-optical instrumentation developed by the Müller lab. The training plan proposed in this application and the results of the project will ser... ## Key facts - **NIH application ID:** 10607448 - **Project number:** 1F32GM149186-01 - **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY - **Principal Investigator:** Petar Nikolov Petrov - **Activity code:** F32 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2023 - **Award amount:** $69,500 - **Award type:** 1 - **Project period:** 2023-01-08 → 2024-01-07 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10607448 ## Citation > US National Institutes of Health, RePORTER application 10607448, Investigating MHC-I downregulation by HIV-1 Nef using cryo-electron tomography with laser-based phase contrast (1F32GM149186-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10607448. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*