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Journal of Virology, March 2008, p. 2405-2417, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.01614-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding{triangledown}

Vineela Chukkapalli,1 Ian B. Hogue,1 Vitaly Boyko,2 Wei-Shau Hu,2 and Akira Ono1*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109,1 HIV Drug Resistance Program, National Cancer Institute, NCI-Frederick, Frederick, Maryland 217022

Received 24 July 2007/ Accepted 13 December 2007

Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P2 depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P2 depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P2-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P2. To examine a putative Gag interaction with PI(4,5)P2, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P2. Using this assay, we observed that PI(4,5)P2 significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P2 for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P2 binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P2-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P2 on the membrane and that the MA basic domain mediates this interaction.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan Medical School, 1150 W. Medical Center Dr., Room 5736A, Ann Arbor, MI 48109. Phone: (734) 615-4407. Fax: (734) 746-3562. E-mail: akiraono{at}umich.edu

{triangledown} Published ahead of print on 19 December 2007.

Journal of Virology, March 2008, p. 2405-2417, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.01614-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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