PROJECT SUMMARY The 5′ untranslated region (5′ leader) of the HIV-1 RNA genome is critical for HIV viral replication. The presence of numerous highly conserved structural elements in this region raises the prospect of novel anti-HIV therapeutics that could bind or disrupt the secondary structure, tertiary folding, and RNA-protein interactions of these elements. Yet, despite decades of investigation of the 5′ leader RNA, there remains substantial controversy over the 5′ leader’s global structure, hindering our basic understanding of the HIV life cycle and delaying our ability to develop curative therapeutics. Previous studies using low-information structural techniques have led to nearly twenty conflicting single-state secondary structure models and numerous long-range tertiary contacts that have been difficult to confirm or refute; indeed, it is possible that the RNA forms numerous distinct biologically important structures. We have recently invented multidimensional chemical mapping approaches that allow unbiased description and dissection of multi-state RNAs as well as RNA-specific cryo-electron microscopy and computational approaches for 3D modeling that we believe can unambiguously resolve these structural controversies. Here, we propose two aims to dissect the complex structural ensemble of the HIV 5′ leader RNA. First, we will bring to bear a recent four-dimensional chemical mapping method called lock-mutate-map-rescue, which employs layers of compensatory mutagenesis and helix-locking mutations to detect, stringently test, and quantitatively establish correlations and anti-correlations between the RNA’s helices and functional elements – information critical to understanding its multi-state structural ensemble. Second, we will resolve long-range tertiary contacts and any stereotyped global 3D conformations in the HIV 5’ leader’s structural ensemble though mutational stabilization, computational modeling, cryo-electron microscopy, and the Multiplexed ·OH Cleavage Analysis tertiary contact mapping method. We will evaluate the success of this multi-state structural description through independent tests involving mutations and protein binding events quantitatively evaluated in vitro; and, later stages, through structure/function experiments, that will test if the HIV 5′ leader transits through the proposed states in cellulo and in virio.