HIV-1 initiates infection by fusing with the target cell membrane through process that is triggered by Env glycoprotein following the engagement of CD4 and coreceptors (CCR5 or CXCR4). At least two host factors, SERINC5 (serine incorporator 5) and IFITM (interferon-induced transmembrane) protein families, are known to incorporate into HIV-1 particles and reduce their infectivity by inhibiting the virus-cell fusion. The mechanisms by which these restriction factors inhibit viral fusion are poorly understood. The main difference in the antiviral activity of these two proteins is the range of restricted viruses. Whereas IFITMs inhibit fusion of many unrelated viruses, SERINC5 is only reported to interfere with retroviral fusion. However, SERINC5 and IFITMs also share striking similarities, including the ability to reduce HIV-1 fusion when expressed in target cells and the varied resistance of HIV-1 strains to these factors that maps to the gp120 variable loop 3. The most accepted model of IFITM-mediated inhibition of fusion, which is supported by our pilot results, is through increasing the membrane stiffness and curvature. Based on the above similarities, the lack of strong evidence for Env-SERINC5 binding and our super-resolution imaging results showing poor colocalization of these molecules, we hypothesize that SERINC5 and IFITMs inhibit HIV-1 fusion by a similar indirect mechanism that involves altering the properties of viral membrane. To test this hypothesis, we will imlpement several cutting-edge techniques to assess the membrane protein and lipid distributions and dynamics on single HIV-1 particles. Specifically, in Aim 1, we will identify SERINC5-resistant Env mutants by deep mutational scanning and characterize these mutants using flow virometry, super-resolution microscopy and correlative cryo-EM. Aim 2 will focus on delineating the effects of SERINC5 on the lipid order and mobility in the viral membrane. In Aim 3, we will investigate whether, like SERINC5, IFITMs alter the architecture and/or dynamics of the HIV-1 membrane. Finally, Aim 4 will test the hypothesis that plasma membrane tension increases, which are mediated by Env-receptor/coreceptor signaling, drive the late stages of HIV-1 fusion at the cell surface and that SERINC5 may inhibit HIV-1 fusion by attenuating these cellular responses. Completion of the proposed Specific Aims will elucidate the mechanism(s) of HIV-1 restriction by unrelated host factors. If these factors inhibit viral fusion through a conserved indirect mechanism involving modulation of membrane properties, and not through direct interactions with viral proteins or cellular receptors, this would be a paradigm-shifting discovery. The results of this project will also delineate the role of cellular signaling and plasma membrane tension in promoting HIV-1 fusion.