Intracellular Dynamics of HIV-Gag: The Role of Calcium and the Activation of Phospholipase C

Abstract

The assembly of the human immunodeficiency virus type 1 occurs at the plasma membrane or within late endosomes/multivesicular bodies, and is determined by the polyprotein Gag. This structural protein is also the only viral component necessary and sufficient for the assembly and the release of virus-like particles (VLPs). It is well known that participation of host cell components is particularly required for any of the many Gag-encoded functions. In particular, as already shown by L.S. Ehrlich et al. 2010, the activation of the phospholipase C and the inositol-(1,4,5)-triphosphate receptor, resulting in an increase of intracellular calcium concentration, are both required for efficient Gag trafficking and VLPs release.So far, it is still unclear whether the Gag protein itself or a cellular factor specifically activates this signaling pathway for the virus release process.We are interested to investigate the activation and the role of the PLC signaling pathway in Gag-expressing cells, and how the intracellular calcium concentration can influence the dynamics of Gag and the VLPs release-process.HeLa cells transfected with Gag fused with a fluorescent protein (e.g. EGFP, EYFP) are our principal tool to investigate the intracellular localization of the viral protein upon activation or inhibition of the PLC signaling pathway. Acetoxymethylester-derivates of fluorescent indicators are used to study variations of the calcium concentration in live cells. Chemical inhibitors and RNAi technology are utilized to turn off the PLC pathway at different steps and time points. Total Internal Reflection Fluorescence microscopy, Forster Resonance Energy Transfer methods and co-immunoprecipitation are applied to study the localization of Gag at the plasma membrane or in the cell-interior, and to identify interactions with specific binding partners of the host cell.