Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain
Authors: Olety Balaji , Veatch Sarah L. , Ono Akira 
Published: May 2015

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 Cδ1 (PH
PLCδ1
), 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

PLCδ
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

PLCδ
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

PLCδ
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

PLCδ
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

PLCδ
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.

IMPORTANCE
Binding 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

PLCδ
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

PLCδ
1

and Gag similarly, whereas PI(4,5)P
2
with saturated acyl chains either recruited PH

PLCδ
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 virion production, the PI(4,5)P
2
acyl chain property may serve as a potential target for anti-HIV therapeutic strategies.