Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

MATIAS OSTROWSKI

Bethesda

Congreso; Meeting of the International Society of Extracellular Vesicles; 2015

ISEV

Introduction: During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and directs HIV-1 assembly. The cellular mechanisms underlying PI(4,5)P2 synthesis at the site of HIV assembly are not known. We hypothesized that exosome secretion and HIV-1 assembly are functionally coupled, and therefore, we undertook the analysis of the role played by Rab27a in HIV-1 assembly. Methods: Rab27a expression was silenced in Jurkat cells as well as in primary CD4+ T lymphocytes and macrophages. Cells were subsequently infected with HIV-1, and viral production was analyzed by ELISA. Alterations in exosome secretion were analyzed by immunoblot. Modifications in endosome trafficking and Gag distribution were analyzed by confocal microscopy. Results: We show that Rab27a controls the trafficking of multivesicular endosomes (MVEs) carrying phosphatidylinositol 4-kinase type 2 alpha (PI4KIIa) towards the PM of CD4+ T cells, promoting exosome secretion, high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2. This phosphoinositide subsequently promotes Gag recruitment and HIV-1 assembly. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. Summary/conclusion: We conclude that by directing the trafficking of PI4KIIa-positive endosomes towards the PM, Rab27a controls exosome secretion and PI (4,5)P2 production at the site of HIV-1 assembly.