Abstract – Project 1 Approximately 40 million people worldwide are living with HIV/AIDS; however, a protective vaccine or functional cure remain elusive despite four decades of intense research. HIV-1 evades the immune system through its rapid structural evolution during infection and replication, and the implications of these structural changes in the HIV-1 Envelope (Env) protein on HIV-1 fusion and entry into cells is not completely understood. Our overall goal in this project is to define the steps leading to formation of the so called pre-hairpin intermediate of the HIV-1 Env to its decay into later fusion intermediates. Initiation of this event is typically described as a ‘harpoon-like” insertion of the FP into the host membrane. While this depiction is illustrative of the earliest anchoring event, it is not physically descriptive. The inherent instability of the pre-hairpin intermediate has rendered observation of its structure intractable. In this project, we aim to combine advances in molecular simulation with cutting-edge experimental methods to interrogate this process at high spatial and temporal resolution. To accomplish these aims, Project 1 will leverage expertise in the structural biology and computational biology cores to develop and apply methods to this problem. Our long-term goal is to develop a complete, time resolved and atomically detailed mechanism of HIV-1 Env fusion. The overall objective for this project is to develop and apply new methods in computational and structural biology to make possible the interrogation of rare transient states along the fusion pathway. The specific targets in this proposal are (i) to determine the conformational transitions that trigger fusion peptide release from its association with the Env trimer, (ii) to examine passage of the fusion peptide from its trimeric anchor to the host membrane surface, (iii) to identify the determinants of fusion peptide insertion, assembly, and anchoring in the host membrane, and iv) to delineate the conformational transitions that facilitate gp120 shedding from gp41. The central hypothesis driving this study is that each transition is tightly regulated, displaying sequential, cooperative control at each stage. The rationale for this project is that advanced, multiscale modelling of this process combined with cutting edge structural biology tools can elucidate the mechanism by which transitions at this intermediate stage of entry are regulated. Upon completion of these aims, we expect to provide kinetically resolved, atomic-level dynamics understanding of HIV-1 Env fusion peptide triggering, release, and membrane engagement. These results will have broad impact on HIV-1 vaccine and drug development specifically and on virus entry investigations generally.