Organization and regulation of intracellular plasma membrane-connected HIV-1 assembly compartments in macrophages

Background: In HIV-1-infected human monocyte-derived macrophages (MDMs), virus particles assemble primarily on intracellularly sequestered plasma membrane domains termed intracellular plasma membrane-connected compartments (IPMCs). Despite their clear role in virus formation, little is known of the organization, composition, dynamics or function of these compartments. Results: We have used amphipathic membrane dyes to reveal the complex three-dimensional structure of IPMCs in whole MDMs and to visualize connections between IPMCs and the cell surface. The observation of similar IPMC structures in both infected and uninfected cells indicates that these compartments are not induced by virus infection, but are present constitutively in MDMs. By expressing a phospholipase C delta pleckstrin homology domain linked to green fluorescent protein, we demonstrate that IPMCs contain phosphatidylinositol 4,5-bisphosphate. Live cell imaging of cells expressing this probe shows that IPMCs are dynamic, but relatively stable, sub-domains of the plasma membrane. As recent electron microscopy studies indicated that portions of IPMCs are coated with beta 2 integrin-containing focal adhesion-like complexes linked to actin, we investigated whether the actin cytoskeleton is required for the organization of IPMCs. In MDMs treated with the actin polymerization inhibitor latrunculin, the normally compact IPMCs dispersed into smaller structures that remained connected to the plasma membrane. Moreover, latrunculin enhanced the release of preformed, mature HIV-1 particles from infected MDMs. Conclusions: IPMCs are constitutive features of MDMs that are continuous with the plasma membrane and are used as unique sites for the assembly of new virions following infection by HIV-1. A functionally intact actin cytoskeleton is required to maintain the organization of the IPMCs and, in HIV-1-infected cells, perturbation of the actin cytoskeleton influences both the organization of the compartment and the release of sequestered virus.