Project Abstract HIV remains a key health challenge, with fundamental aspects of the viral replication machinery remaining poorly understood. Building on our extensive studies of HIV-1 reverse transcriptase (RT) structure and function, we propose to further investigate RT’s roles within the reverse transcription complex (RTC) and the viral maturation process. Our previous work has provided fundamental insights into HIV-1 RT's molecular architecture, including key structures of RT complexed with diverse nucleic acid substrates and nucleoside, non-nucleoside, and other inhibitor types, and has contributed to elucidating molecular mechanisms of inhibition and resistance. We propose to address three critical knowledge gaps: (1) further definition of the specific conformational states and transitions during first- and second-strand DNA synthesis initiation and elongation; (2) the molecular interaction between RT and its partner host protein eEF1A and its functional role within the RTC; and (3) the role of the RT portion of Gag-Pol in HIV virion assembly and maturation. Using cutting-edge cryo-EM and X-ray crystallography, complemented by biochemical and virological studies through established and new collaborations, we will determine structures of RT complexes that capture these dynamic processes. Our preliminary data include novel structures of RT/DNA:RNA polypurine tract complexes, successful engineering of polyprotein constructs enabling the visualization of the RT and protease (PR) dimeric regions within Gag-Pol and Pol polyprotein structures by cryo-EM, and optimization of protein expression and structure determination for studying the RT-eEF1A interaction. Anticipated outcomes and insights include high- resolution structures detailing: 1) reverse transcription initiation phases and transition to elongation; 2) RT's interactions with eEF1A and rationale for its role in promoting reverse transcription; 3) how RT participates in virion assembly within Pol and Gag-Pol