- The proteolytic cleavage of the poly-protein HIV-1 Gag, which is assembled on the surface of plasma membranes of infected cells, drives the conversion of the virus from the initial immature, non-infectious form to the functionally distinct mature, infectious form. Gag cleavage results in a series of structural changes, ultimately leading to the formation of a mature core. Current models for assembly of the mature core suggest that the cleaved HIV capsid protein (CA) nucleates in a concentration-dependent manner, and polymerizes forming the conical core in a diffusion-controlled process. These models also postulate that the core begins to grow at its narrow end, and stops growing once it reaches the membrane at the opposite end. Thus, the size of the virus itself is expected to be the primary factor that determines core size. Our findings challenge this view.Cryo-electron microscopic analyses show that along with infectious viruses, viral isolates also comprise large membranous structures that contain multiple, freely-floating mature cores. Numerous instances of membrane-attached assembly intermediates with partially formed “core-rolls” that are at different stages of conversion from a planar Gag lattice to the mature core are also observed. These results indicate that the mechanism of core formation involves a non-diffusional, cooperative transition triggered by cleavage of the immature Gag lattice, resulting in its rolling away from the plasma membrane to form sheets that wrap around the viral RNA. Unlike the present models, our mechanism predicts that the generation of infectious HIV-1 will be severely affected by incomplete cleavage CA from the Gag matrix (MA), and that even a small percentage of uncleaved MA-CA would prevent the core from rolling away. This prediction explains previous experimental results; only 4% of cleavage-resistant MA-CA is sufficient to cause a 50% reduction of infectivity.