This application for the Center for Structural Biology of HIV RNA (CRNA) focuses on determining the structural and mechanistic bases of HIV-1 RNA-related replication functions and the host’s response at the cellular, viral, and atomic levels. Although considerable progress has been made over the past 40 years in understanding how proteins function in HIV-1 replication, comparatively little is known about how HIV-1 RNA structures, dynamics, trafficking, and interactions with proteins alternately enable or limit virus replication. The past decade has provided transformative examples of the therapeutic use of RNA and RNA-targeted therapies, and recent progress by members of the proposed team has begun to unlock structural and dynamic features of RNA elements in HIV-1. Because HIV-1 RNA is exceptionally rich in biologic functions, the potential for RNA-targeted approaches in the prevention, maintenance, and cure of HIV-1 disease is highly compelling. However, this potential is limited by the general paucity of high-resolution structural information for RNA and protein-RNA complexes, which reflects inherent challenges in using RNA as a subject for structural analysis and the inadequacy of traditional biophysical approaches to address these challenges. The CRNA will face these challenges for HIV-1 RNA with its multidisciplinary team of structural biologists, chemists, cell and computational biologists, biochemists, immunologists, and virologists. Many are leaders in the study of HIV-1 RNA and the roles of its structures in virus replication, while others are new to the HIV-1 field and bring a fresh perspective and complementary expertise. Together, this team brings cutting edge technologies and incisive biologic approaches to overcome current technological obstacles, enabling mechanistic determination of the role of HIV- 1 RNA structures and associated proteins in viral transcription, splicing, translation, packaging, particle assembly, and interactions with host factors. These studies will enable the CRNA to advance goals of clinical relevance, including the development new RNA-centered strategies for treating HIV-1, the reactivation of latent proviruses as a potential approach for eradicating HIV-1 infection, and the augmentation of host defenses against HIV-1 infection. The proposed studies will also result in the development of powerful new computational, imaging, and structural biology technologies that can be applied to all areas of RNA biology.