ESCRT machinery potentiates HIV-1 utilization of the PI(4,5)P(2)-PLC-IP3R-Ca(2+) signaling cascade |
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Authors: | Ehrlich Lorna S Medina Gisselle N Carter Carol A |
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Affiliation: | Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA |
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Abstract: | Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P7TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY36PXnL motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that “gates” Ca2+ release from intracellular stores, triggers Ca2+ cell influx and thereby functions as a major regulator of Ca2+ signaling. In the present study, we determined whether the L domain links Gag to Ca2+ signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca2+ was elevated, events indicative of induction of Ca2+ signaling. The results suggest that L domain function, ESCRT machinery and Ca2+ signaling are linked events in Gag release. |
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Keywords: | HIV-1, human immunodeficiency virus type 1 IP3R, inositol 1,4,5-triphosphate receptor PLC, phospholipase C IP3, inositol (1,4,5)-triphosphate VLP, virus-like particle ER, endoplasmic reticulum DAG, diacylglycerol PI(4,5)P2, phosphatidylinositol-(4,5)-bisphosphate SOCE, store-operated calcium entry WT, wild type m-3M3-FBS, 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)benzenesulfonamide 2-APB, 2-aminoethoxydiphenyl borate GFP, green fluorescent protein DMEM, Dulbecco's modified Eagle's medium DMSO, dimethyl sulfoxide siRNA, small interfering RNA |
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