Structural Analysis of HIV-1 Maturation Using Cryo-Electron Tomography

Abstract

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA. HIV-1 buds from the plasma membrane of infected cells in an immature form with the polyprotein Gag as its major component. Maturation into an infectious form requires cleavage of Gag in five positions. This process is an important target for antiretroviral drugs. Here we studied changes in the structure of the virus that occur during maturation, making use of virus variants in which different combinations of cleavage sites were mutated to prevent cleavage at those sites. We used cryo-electron tomography and sub-tomogram averaging to visualise the arrangement of Gag in 3D. We show that the fastest cleavage event leads to condensation of the RNA genome complexed with viral proteins. This inner RNA/protein structure appears to maintain a link with the remaining Gag lattice. Processing on both sides of CA-SP1, the main structural module of Gag, is required for disassembly of the immature Gag lattice, while removal of SP1 is needed in addition for mature core formation. The results provide structural correlates of the ordered processing events during HIV-1 maturation and shed light on the mechanism of action of bevirimat, an inhibitor of CA-SP1 cleavage in clinical trials.