Project Summary/Abstract The overall goal of this project is to understand how HIV-1 packages its RNA genome. During virus assembly, two copies of the viral RNA are trafficked to the plasma membrane and anchored to assembly sites by a small number of viral Gag polyproteins (~24 or fewer). The resulting complex functions as a nucleant for further virus assembly, recruiting additional Gag molecules and promoting budding. Although mechanistic and atomic level details are unknown, studies suggest that assembly of the Gag:RNA complex is mediated by a combination of intermolecular interactions between the capsid (CA) domains of Gag, and between Gag’s nucleocapsid domain (NC) and an RNA packaging signal (ΨCES) located within the 5′-untranslated region of the genome. The proposed studies aim to (i) identify the structural determinants and mechanism of Gag assembly on the viral RNA packaging signal during different stages of assembly (supported by the K99). Additionally, the proposal aims to (ii) collect more structural information of host cell factor interaction, specifically Staufen1, with the viral RNA (R00), raising potential novel target sites for drug development. The K99 research, conducted under the mentorship of Dr. Michael F. Summers (primary mentor at UMBC) and Dr. Owen Pornillos (secondary mentor at the University of Utah), will employ an integrated structural biology approach, where isotope-edited nuclear magnetic resonance spectroscopy (NMR) will be used to determine key intermolecular Gag:Gag and Gag:RNA interactions that promote assembly, and Cryogenic Electron Microscopy to visualize atomic-level details of the Gag:RNA complex that nucleates virus assembly. Mechanistic hypotheses will be tested by in-cell studies under the guidance of virologist Dr. Alice Telesnitsky (member of the advisory committee at University of Michigan). Expertise developed in NMR, Cryogenic Electron microscopy, and cellular techniques will prepare me for an independent career to work within the broader area of host-virus RNP interactions. Overall, these studies will provide the first structural and mechanistic insights for the ribonucleoprotein complex that nucleates HIV-1 assembly and its interactions with essential host factors and will provide Dr. Hollmann skills to establish an independent and inclusive research program.