Calcium-Mediated Fusion between Endo-Lysosomal Compartments Enhances Virus-Like Particles Release

Abstract

Assembly of the human immunodeficiency virus (HIV) is governed by the structural polyprotein Gag, which is necessary and sufficient for the release of virus-like particles (VLPs) from the host cell. Although the plasma membrane has been recognized as the major site for the production of VLPs, in some cell-types Gag is targeted to late endosomes/multivesicular bodies (LE/MVBs), where assembly and budding take place. Virus release into the extracellular space then occurs after regulated exocytosis. It is well accepted that the release of VLPs requires participation of different host cell components. In particular, it was shown that induction of a transient rise in cytoplasmic Ca2+ increased the amounts of VLPs in MVBs, and resulted in a dramatic enhancement of VLPs release (Perlman M. et al., 2006). However, although cellular factors have been already proposed as mediators of Ca2+ provision (Ehrlich L. et al., 2010), how Ca2+ can promote the release of VLPs remains to be determined.With FACS analyses on live cells, we could identify variations of intracellular Ca2+ in Gag-expressing cells treated with Ca2+ fluorescent indicators. High-resolution confocal and electron microscopy have confirmed that Gag can assemble and bud into VLPs in lysosomes (Ly) and LE. Furthermore, we could show for the first time that Ca2+ released specifically from those compartments causes formation of Ly/LE hybrid organelles, which in turn fuse with the PM and release VLPs into the extracellular space. This heterotypic fusion process requires components of the SNARE complex and the Ca2+ sensor protein Synaptotagmin VII, which regulates Ly exocytosis. All these elements constitute a productive pathway for virus assembly and release. We believe that the Gag protein itself, or a cellular factor recruited by Gag, might promote the increase of Ca2+ required for this process to function.