PROJECT SUMMARY/ABSTRACT The goal of the proposed studies is to understand how HIV-1 sequence diversity impacts viral genome dimerization, a requirement for viral replication. Like nearly all retroviruses, HIV-1 selectively packages two copies of its full-length genome after the formation of a dimer – a process essential not only to packaging, but also reverse transcription and recombination. The sponsor’s lab took a pioneering role in the structural characterization of the highly conserved HIV-1 dimeric 5′-leader, the region believed to initiate genome dimerization. Several outstanding questions remain regarding the mechanism and structures involved in the process. Current proposals support a two-step mechanism beginning with a kissing-loop interface that then matures into a more extensive intermolecular interface. While in vitro studies support this mechanism, most are isolated to RNA fragments or ignore important cellular/viral factors. Studies of HIV genome dimerization are also complicated by the enormous genome plasticity of HIV-1 that is driven by mutation and recombination, leading to sequence diversity within the dimer interface. This diversity stratifies strains into two dimer classes, those that are thermodynamically stable (nonlabile strains) and those that readily dissociate (labile strains). I will characterize the differences between these two dimer classes by studying two model HIV-1 strains in the context of the intact dimeric interface in solution, in cells, and in viruses: NL4-3 (nonlabile) and MAL (labile). Current methods to study labile dimers are limited, as they readily dissociate in native gel electrophoresis assays; therefore, suggesting a need to rely upon methods that will assess the equilibrium, solution state for each strain. We will begin with a biophysical characterization of both strains, specifically characterizing the thermodynamics, kinetics, and structures of the dimeric interface using Fluorescence Correlation Spectroscopy (FCS) and Nuclear Magnetic Resonance (NMR) (Aim 1). Preliminary FCS data has shown our ability to monitor dimer formation at concentrations and timescales previously inaccessible. Preliminary data using our 2H-edited NMR approach suggest we can directly probe for intermolecular interactions in the full-length, dimeric HIV-1 MAL 5′-leader (>230 kDa), allowing us direct comparison with the previously characterized NL4-3 extended dimer. I will also compare the dimerization process of these two strains in cells and viruses (Aim 2). We have now collected initial in vitro data validating our novel fluorescent labeling strategy to discriminate intermolecular and intramolecular RNA interactions that can now be applied in the context of viral replication in cells. We hypothesize that labile dimers exhibit primarily a kissing dimer structure throughout assembly, highlighting a higher stability to the kissing dimer than was previously thought, as well as implying the existence of a strain specific dim...