Abstract: Through infection of CD4 positive (CD4+) T lymphocytes, the Human Immunodeficiency Virus type 1 (HIV-1) has claimed twenty-five million lives since its discovery in 1983. Although it has been well established that HIV-1 initiates a T cell infection by binding to CD4 and chemokine coreceptors on the cell surface, the early events in HIV-1 infection of CD4+ T cells remain poorly understood. A variety of different techniques have been used over the years to study mechanisms of HIV-1 entry. However, one technical limitation that is inherent in all these methods is the inability to track the fate of a single HIV-1 virion from the very beginning of viral entry to chromosomal integration. In all these experiments, a population of viruses and cells were incubated and measured. Because each entry event can lead to proviral DNA integration with a finite probability, it is therefore difficult to establish a causative link between entry pathway and productive infection. The goal of this project is to develop a set of nanoscopic and novel technologies that we can harness to define the pathway and interactions by which HIV-1 infects CD4+ T cells. We are developing a technique based on optical tweezers that can manipulate a single HIV-1 virion, deliver it to CD4+ T cell and thus allows us to determine the fate of CD4+ T cell upon entry by a single virion. This technique will allow us to unambiguously define the molecular mechanisms of HIV-1 infection. Furthermore, we propose to measure directly the interactions between a single HIV-1 virion and receptors in the context of a live T cell. Collectively, these studies will contribute to a definitive and quantitative understanding of early events in HIV infection, which may help therapeutic development that is aimed to block HIV-1 entry to CD4+ T cells. The techniques developed herein can be useful for studying cellular uptake of not only viruses but other molecules, macromolecular assemblies and nanoparticles, and are applicable to a wide-range of ligand-receptor interactions on the cell surface. Public Health Relevance: The early events in HIV infection of CD4+ T cells are poorly understood. This proposal aims to develop a set of nanoscopic and other novel techniques to study HIV infection of CD4+ T cells in real time, one virion at a time. If successful, the results from this study will establish for the first time a causative link between HIV entry pathway and the productive infection of CD4+ T cells, which will help therapeutic development that is aimed to block HIV-1 entry to CD4+ T cells.