ABSTRACT The HIV/AIDS pandemic remains a global burden on human health.1 Although antiretroviral therapy (ART) reduces deaths from HIV infection, prophylactic vaccine(s) and a functional cure for the millions infected are lacking.2 Success in these areas will require improved understanding of HIV transmission and dissemination and the mechanism of CD4 T cell depletion, which is the hallmark of immune deficiency.3 In addition, developing antiviral therapies and vaccines against HIV envelope (Env), the only viral protein on the surface of the HIV virion4,5, requires understanding its conformational dynamics and how it is activated for virus–host cell fusion. We propose to fill these knowledge gaps through imaging experiments that will: 1) Visualize the dissemination of HIV-1 following transmission in humanized mouse models and non-human primates6-9, 2) Determine the cause of CD4 depletion and visualize cellular events underlying viral restriction, 3) Delineate how the HIV Env trimer is activated for fusion10,11, and 4) Define how Env is inhibited by host cell restriction proteins of the SERINC family.12-14 This approach will reveal bottlenecks in virus dissemination, mechanisms of restriction, and vulnerabilities in activating HIV Env that can be exploited therapeutically.