The highly conserved 5' untranslated region (5'UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5'UTR can transition between at least two conformational states. In one state the genome remains a monomer， as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state， the DIS is exposed， and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5'UTR determines the fate of the genome. In this work， the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5'UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNA， the primer for initiation of reverse transcription， can promote the kissing dimer but not the extended dimer. In contrast， HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNA Our data are consistent with an ordered series of events that involves primer annealing， genome dimerization， and virion assembly.