7. Project Summary/Abstract The overall goal of this project is to understand how the HIV-1 5′-leader RNA differentially directs diverse functions during viral replication, including intracellular trafficking, initiation of translation, recruitment of viral structural proteins (called Gag), and nucleation of virus assembly. Proposed studies build on our recent discovery that HIV-1 RNAs are transcribed with one, two or three 5ʹ-guanosines via alternate (heterogeneous) transcriptional start site usage – a new paradigm in HIV-1 RNA biology. Our studies revealed that 5′-capped transcripts that begin with a single guanosine (Cap1G) adopt a structure that sequesters the 5′-cap, promotes dimerization, and exposes Gag binding sites, thereby promoting their functions as genomes (gRNA). In contrast, 5′-capped transcripts that begin with two or three guanosines (Cap2G/Cap3G) adopt monomeric structures with an exposed 5′-cap that function in splicing and translation (mRNAs). The central hypothesis guiding our proposed studies is that cap sequestration enables the HIV-1 gRNA to avoid capture by the cellular RNA processing and translation machinery, and that a small number of Gag proteins (~2 dozen) are cooperatively recruited to assembly sites on a well-defined packaging signal (ΨCES) located within the gRNA 5′- leader. We now aim to identify molecular determinants of alternate transcription initiation and exploit this knowledge to differentially examine HIV-1 mRNA and gRNA trafficking and interactions in cells, probe RNA structural changes that occur during virus assembly and maturation, and identify key interactions that promote Gag:RNA assembly and might be exploited for drug targeting. Preliminary NMR and EM findings suggest that it should now be possible to determine the 3D structure of the Gag:RNA complex that nucleates HIV-1 virus particle assembly. NMR studies of large RNAs and protein-RNA complexes are technically challenging – the average size of NMR-derived RNA structures in the RNA Structure Database is only 30 nucleotides. But the potential payoff of the proposed studies is substantial and could ultimately lead not only to a more detailed understanding of how HIV-1 replicates, but also to the development of new approaches for the treatment of AIDS and to the engineering of gene delivery vectors with improved packaging efficiencies.