During the late phase of HIV-1 infection cycle, the virally encoded Gag polyproteins are targeted to the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag–PM binding is mediated by interactions of the N-terminally myristoylated matrix (MA) domain with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Concurrent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Env recruitment, and hence gp41, to a nascent virion is essential for downstream infectivity. Without gp41, there is no fusion and no infectivity. Several lines of evidence suggest that Env incorporation is mediated by interactions between the cytoplasmic tail of gp41 (gp41CT) and the MA domain of Gag. It has been long recognized that only a few (< 10) gp41 molecules are embedded in the MA layer. Both gp41CT and a well- formed MA lattice are essential for incorporation and infectivity. It appears that it is not sufficient to only embed gp41CT in the MA layer, but it is necessary for the MA layer to undergo a cleavage induced maturation step for gp41 to become fully active. The incorporation and activation of gp41 has been a long-standing problem whose solution requires a structural approach. Recent low-resolution cryo-electron tomography (cryo-ET) studies proposed a model in which the MA domain undergoes a structural transformation to form distinct MA lattices during assembly and upon maturation. During the current funding period, our lab has shown that MA forms a hexamer of trimers lattice with a central hole, thought to accommodate gp41CT to promote incorporation into virions. We have also shown that PI(4,5)P2 is capable of binding to alternate sites on MA, consistent with a novel and perhaps distinct MA–membrane binding mechanisms during assembly of the immature particle and upon maturation. However, the structural details of the MA lattice (immature and mature) bound to membrane, factors that govern the MA conformational switch, factors that (de)stabilize the MA lattice, and the structural basis for MA–gp41CT interaction during assembly and upon maturation, are still lacking. The aims of this proposal are designed to elucidate the molecular mechanisms that render HIV infectious by studying how gp41CT is embedded in the MA layer. We devised innovative “controlled assembly” approaches which will enable us to generate biologically authentic substructures of the immature and mature gp41CT–MA complex. To study these substructures, we have developed cryo-electron microscopy (cryo-EM) approaches which will allow us to use single particle rather than cryo-ET techniques to enable near atomic structural determination. Our aims are to (1) determine the structural basis for MA lattice formation during assembly of the immature particle and upon maturation, (2) determine factors critical for MA lattice formation and Env incorporation, and to (3) determine the structure of the MA–gp41CT–membrane complex ...