Project 2 Summary HIV-1 RNA interacts with many host proteins, some of which enhance and some of which inhibit viral replication. This project will focus on protein:RNA complexes involving HIV-1 RNA and host proteins. Because the HIV-1 genome has all the features of a cellular mRNA, the question of whether and how the HIV-1 genome is differentiated from cellular mRNAs, or ‘sensed’ by host cell proteins is an important principle underpinning this project. Distinguishing features of HIV-1 RNAs include RNA sequence, structures, compositional biases and base modifications. These features of the HIV-1 RNA can enable recognition by antiviral proteins or sensors, which feature prominently in the project. The team will continue its existing collaboration on the structure and function of APOBEC3 proteins, and will build on their prior crystallographic studies of APOBEC3H:RNA interactions to determine how APOBEC3H:RNA binding inhibits HIV-1 infection independently of deamination activity. The team will also determine the structure of an APOBEC3H-Vif complex and will use structural approaches to determine how viral RNA is recognized by additional APOBEC3 proteins. Additionally, the team will build on its prior groundbreaking discoveries of zinc finger antiviral protein (ZAP) and its CpG RNA target sequence to explore the function of protein complexes containing ZAP and its cofactors. They will determine the structures of ZAP domains, the full-length ZAP protein, and complexes containing ZAP with cofactors TRIM25, Riplet and/or KHNYN. They will also use structural biology approaches to determine the mechanistic basis for specific RNA recognition by ZAP, and biological assays to determine the features of optimal CpG-rich ZAP target sequences. The team will also pursue structural, biochemical, and biological investigations of another antiviral protein that binds HIV-1 RNA, TRIM56. The mechanism of action of TRIM56 is poorly understood, thus the team will determine which viral and cellular RNAs are recognized by TRIM56, and how that recognition leads to recruitment of effector functions that affect the fate of targeted RNAs and consequently HIV-1 replication. In addition to detailed studies of known HIV-1 RNA:host protein interactions, the team will also execute a number of exploratory/discovery studies, including various screening approaches to identify cellular proteins that interact with HIV-1 RNA. They will identify novel host proteins that participate in the epitranscriptomic regulation of HIV- 1 RNA fate and investigate the how the m6A reader YTHDF2 regulates RNA stability. The team will also identify RNA-binding interferon-stimulated gene products with anti-HIV-1 activity and zinc finger proteins that affect HIV- 1 replication Finally, the team will explore how the unusually biased HIV-1 RNA nucleotide composition affects viral replication. Thus, the scope of the project encompasses investigations intended to discover novel RNA- protein interactions, as ...