Recombinant HIV-1 Gag protein, purified from bacteria, is a soluble protein, but assembles into virus-like particles (VLPs) in a defined in vitro system upon addition of nucleic acid (NA). We have tried to understand how NA-binding promotes assembly. Experiments with Gag-leucine zipper chimeric proteins suggest that when Gag attains a high local concentration, it undergoes a change which primes it for assembly; cooperative NA-binding would be one way it could reach a high local concentration. We have focused on the SP1 region of Gag, which lies between the CA domain (principally responsible for Gag-Gag interactions in VLPs) and the NC domain (principally responsible for NA-binding). Many subtle mutations in SP1 cause drastic disruption of VLP assembly. If the SP1 sequence were folded into an α-helix, the helix would be amphipathic, with a polar face and a hydrophobic face. We have found that a peptide representing SP1 folds into a helix when it is at high concentration; presumably, at these concentrations, helices can aggregate and bury their hydrophobic faces within helix bundles. We now report that small proteins consisting of SP1 fused to a dimerizing leucine zipper form discrete tetramers in solution. However, many mutations in these SP-zipper chimeras cause them to form dimers rather than tetramers; therefore, SP1-SP1 interactions are responsible for the association of the zipper-induced dimers into tetramers. As these specific mutations also disrupt assembly by Gag, the same SP1-SP1 interactions are also significant in VLP assembly. We hypothesize that folding of SP1 into a helix primes Gag for VLP assembly, perhaps by exposing new interfaces in the CA domain. Information on the structure of these SP1 bundles will be presented.