OVERALL Antiretroviral therapy has turned HIV/AIDS into a chronic disease, yet the emergence of drug-resistant variants and comorbidities after long-term ART remain a concern. Therefore, alternative approaches to inhibit infection and cure AIDS are needed. The proposed “Pittsburgh Center for HIV Protein Interactions” (U54 PCHPI) is well- positioned to succeed in the search for the much-needed alternative targets for HIV-1 suppression. The U54 PCHPI is a highly integrated, collaborative effort, building on established productive collaborations. The fundamental research program will focus on structurally characterizing HIV-1 protein and protein-nucleic acid complexes involved in three aspects of infection: HIV-1 assembly and maturation, ingress and nuclear entry, and integration. Studies to address these stages of the infection cycle will be carried out in three projects, each focused on one of these areas, and in four scientific cores (Computational, Cryo-EM/ET, NMR and Virology) along with Administrative and Developmental cores. Importantly, our program will work to define the structural basis underlying maturation and allosteric integrase inhibitor activities to promote a mechanistic understanding and seeks to identify paths of resistance development. Further, we will identify new targets for inhibition of HIV- 1 by defining interaction interfaces within capsid-host protein/nucleic acid complexes involved in trafficking, nuclear entry, and integration, with a particular focus on native pre-integration complexes (PICs). We will also develop tools for examining capsid interactions under near-native conditions (in situ NMR spectroscopy and single-molecule CLEM for HIV-1). In addition, a robust management plan, implemented via an Administrative Core, will ensure a cohesive effort with frequent and transparent communications, while our mission to facilitate research career development will be enabled by a collaborative development program, a mentoring program, and a researcher embedding program, among other initiatives in the Developmental Core. Upon completion of our aims, we expect to have identified and characterized, at high resolution, several previously unknown/uncharacterized interaction interfaces in HIV-1 protein complexes, alone and with inhibitors, between HIV-1 RNA and proteins, and within PIC components, including retroviral intasome interactions with host chromatin. Detailed knowledge of such interfaces will enable structure-guided improvements in inhibitor design as well as identify potential new targets for inhibition.