Genome dimerization is a conserved feature of retroviral replication and a critical step in the HIV-1 life cycle, but how it is regulated is incompletely understood. Here, we developed FARS-seq (Functional Analysis of RNA Structure) to comprehensively identify sequences and structures within the HIV-1 5 UTR influencing dimerization. We found nucleotides important for dimerization throughout the HIV-1 5 UTR and identified distinct structural conformations in monomeric and dimeric RNA. The dimer displayed TAR, PolyA, PBS, and SL1-SL3 as stem-loops. In the monomer, SL1 was dramatically reconfigured into long- and short-range base-pairings with polyA and PBS, respectively. The polyA-SL1 interaction disrupts the major packaging motifs, and the PBS-SL1 interaction functionally couples the primer binding site with dimerization, Pr55Gag binding and packaging. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.
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