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To establish a new infection and gain entry to a cell, Human Immunodeficiency Virus (HIV) must fuse its lipid envelope with the host cell plasma membrane. This process of HIV membrane fusion is catalyzed by conformational rearrangements of the HIV surface glycoprotein, Env, and proceeds through a series of intermediate states involving rearrangements of the host and viral membranes. We sought to better understand HIV membrane fusion and the mechanism by which it can be blocked by restriction factors Serinc3 and Serinc5. To do so required the development of new assays and tools. We demonstrated that plasma membrane blebs incorporating the viral receptor, CD4, and co-receptor CCR5, were useful model membranes for studying HIV membrane fusion by both total internal reflection fluorescence microscopy (TIRFm) and cryo electron tomography (cryoET) (Chapter 2). Additionally, we designed a modular perfusion chamber that improved reliability and throughput of the TIRF-based fusion assay (Appendix B). With these, we characterized Serinc-disrupted and unperturbed HIV membrane fusion intermediates and showed that Serinc5 incorporation into HIV particles obstructs fusion at multiple intermediate steps. Next, to better understand the mechanism by which Serinc5 disrupts fusion, we examined the importance of the viral membrane for Serinc5-mediated inhibition of HIV membrane fusion (Chapter 3). We found that pretreatment of HIV particles with phosphatidylethanolamine (PE) selectively increased fusion of Serinc5-containing HIV particles regardless of acyl chain length or saturation, demonstrating the importance of lipid headgroup interactions for Serinc5 restriction. Crucially, we demonstrated that Serinc5 increases the fraction of the viral membrane in an ordered lipid phase by two methods and provided the first examination of lipid phase behavior in an intact viral membrane. Based on these data, we describe a model for the mechanism of Serinc5-mediated restriction of HIV membrane fusion. In addition to the assays and tools listed above, we developed a method to construct semi-supported lipid bilayers (SSLBs) on holey carbon supports for cryoEM and showed that SSLBs can reproduce multiple features of conventionally prepared supported lipid bilayers (Chapter 4). We then demonstrated applications of cryoEM of SSLBs for studying protein oligomerization, assembly, and interactions with membranes at spatial frequencies not accessible by light microscopy. The present work provides a detailed view of HIV fusion and how it is disrupted by Serinc5’s alteration of the viral membrane. Additionally, we describe tools for the study of membrane processes, including fusion.