Gag Localization and Virus-Like Particle Release Mediated by the Matrix Domain of Human T-Lymphotropic Virus Type 1 Gag Are Less Dependent on Phosphatidylinositol-(4,5)-Bisphosphate than Those Mediated by the Matrix Domain of HIV-1 Gag

Murine leukemia virus (MLV) can efficiently spread in tissue cultures by polarizing assembly to virological synapses. The viral envelope glycoprotein (Env) establishes cell-cell contacts and subsequently recruits Gag by a process that depends on its cytoplasmic tail. MLV Gag is recruited to virological synapses through the matrix domain (MA) (J. Jin, F. Li, and W. Mothes, J. Virol. 85:7672-7682, 2011). However, how MA targets Gag to sites of cell-cell contact remains unknown. Here we report that basic residues within MA are critical for directing MLV Gag to virological synapses. Alternative membrane targeting domains (MTDs) containing multiple basic residues can efficiently substitute MA to direct polarized assembly. Similarly, mutations in the polybasic cluster of MA that disrupt Gag polarization can be rescued by N-terminal addition of MTDs containing basic residues. MTDs containing basic residues alone fail to be targeted to the virological synapse. Systematic deletion experiments reveal that domains within Gag known to mediate Gag multimerization are also required. Thus, our data predict the existence of a specific "acidic" interface at virological synapses that mediates the recruitment of MLV Gag via the basic cluster of MA and Gag multimerization.

Section: Discussionmentioning

confidence: 99%

Basic Residues in the Matrix Domain and Multimerization Target Murine Leukemia Virus Gag to the Virological Synapse

Jin

Herrmann

et al. 2013

“…We previously demonstrated that binding of HIV-1 Gag to liposomes containing a 2:1 ratio of POPC and POPS requires the presence of PI(4,5)P 2 lipids (3,18,20). If Gag is treated with RNase, however, POPS mediates the efficient binding of Gag to liposomes lacking PI(4,5)P 2 (18).…”

Section: Efficient Binding Of Hiv-1 Gag-yfp To Guvs Requires Treatmenmentioning

confidence: 99%

“…RNA bound to MA inhibits binding of Gag to acidic lipid-containing liposomes, such as those composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPS (18)(19)(20). Such inhibition is observed with both in vitro-synthesized Gag (18)(19)(20) and Gag expressed in cells (21).…”

mentioning

confidence: 99%

Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain

Olety

Veatch

Ono

2015

Self Cite

HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P 2 -specific pleckstrin homology domain of phospholipase C␦1 (PH PLC␦1 ), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P 2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P 2 with unsaturated or saturated acyl chains affect membrane binding of PH PLC␦ 1 and Gag. Both unsaturated dioleoyl-PI(4,5)P 2 [DO-PI(4,5)P 2 ] and saturated dipalmitoyl-PI(4,5)P 2 [DP-PI(4,5)P 2 ] successfully recruited PH PLC␦ 1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P 2 but not DP-PI(4,5)P 2 recruited Gag to GUVs, indicating that PI(4,5)P 2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P 2 , cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PH PLC␦ 1 regardless of PI(4,5)P 2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P 2 had DO-acyl chains. DP-PI(4,5)P 2 -containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P 2 still promoted recruitment of Gag, but not PH PLC␦ 1 , to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P 2 acyl chains in membrane binding of two PI(4,5)P 2 -binding proteins, Gag and PH PLC␦ 1 . Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P 2 containing an unsaturated acyl chain over DP-PI(4,5)P 2 , suggesting that Gag sensitivity to PI(4,5)P 2 acyl chain saturation is determined directly by the matrix-PI(4,5)P 2 interaction, rather than indirectly by a myristate-dependent mechanism. IMPORTANCEBinding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P 2 . Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P 2 -interacting domains represented by PH PLC␦ 1 . However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P 2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P 2 with an unsaturated acyl chain recruited PH PLC␦ 1 and Gag similarly, whereas PI(4,5)P 2 with saturated acyl chains either recruited PH PLC␦ 1 but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P 2 acyl chains differentially modulate membrane binding of PI(4,5)P 2 -binding proteins. Since Gag membrane binding is essential for progeny...

“…Even at higher concentrations (ϳ50%), palmitoyl-oleoyl-PS (POPS), the most abundant form of PS in viral and plasma membranes (21), does not support efficient liposome binding although di-oleoyl-PS does (20). In previous studies (9,22), we observed that RNase A treatment drastically increases binding of Gag to liposomes with a 2:1 ratio of POPC and POPS (here termed PCϩPS liposome). To determine the minimal RNA concentration that is sufficient to inhibit membrane binding of Gag, we added back different amounts of RNA to RNase-treated Gag.…”

mentioning

confidence: 99%

Evidence in Support of RNA-Mediated Inhibition of Phosphatidylserine-Dependent HIV-1 Gag Membrane Binding in Cells

Chukkapalli

Inlora

Todd

et al. 2013

Self Cite

The matrix domain promotes plasma-membrane-specific binding of HIV-1 Gag through interaction with an acidic lipid phosphatidylinositol-(4,5)-bisphosphate. In in vitro systems, matrix-bound RNA suppresses Gag interactions with phosphatidylserine, an acidic lipid prevalent in various cytoplasmic membranes, thereby enhancing the lipid specificity of the matrix domain. Here we provide in vitro and cell-based evidence supporting the idea that this RNA-mediated suppression occurs in cells and hence is a physiologically relevant mechanism that prevents Gag from binding promiscuously to phosphatidylserine-containing membranes.