Retroviruses are enveloped viruses that deliver their RNA genome into the cytoplasm of the host by membrane fusion and uncoating of the viral capsid. Despite the importance of these events for the establishment of viral infection, little is known about the molecular mechanism. The Avian Leukosis virus (ALV) and Murine Leukemia viruses (MLV) provide excellent model systems to study these processes. Previously, both viruses were believed to enter cells by a pH-independent process at the plasma membrane. However, our recent studies provide compelling evidence that ALV is first internalized to reach an acidic intracellular compartment where fusion is induced by low pH. A number of important questions must now be addressed. First, from which intracellular compartment does ALV enter the cytoplasm? Secondly, two surface receptors for subgroup A of ALV have been identified. Both allow efficient viral entry but differ in their internalization mechanisms. How do both receptors mediate ALV entry? Do both entry pathways merge to reach a common low pH compartment or are there several that can be utilized for viral entry? Third, where do pH-independent retroviruses such as the human immunodeficiency virus (HIV) and MLV enter cells? Do these viruses enter at the plasma membrane or are they internalized by endocytosis despite their pH-independence? Finally, how do membrane fusion and uncoating of the viral capsid lead to the delivery of the viral genome into the cytoplasm? Do both events proceed simultaneously or are they timely resolved and biochemically distinct? The goal of this proposal is to identify the cellular compartments through which both viruses enter the cytoplasm and to understand whether uncoating of retroviral capsids is directly linked to membrane fusion or proceeds as a distinct reaction in the cytoplasm. Toward this end we will apply a cell biological approach that integrates fluorescence imaging in living cells with functional assays and biochemical techniques. Specifically, we will: 1) determine endocytic internalization routes of ALV as a function of two different receptors, 2) identify cellular compartments through which the pH-independent viruses HIV and MLV enter cells, and 3) dissect fusion and uncoating reactions during viral entry.