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Journal of Virology, April 2003, p. 4805-4817, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4805-4817.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Human Immunodeficiency Virus Type 1 Assembly and Lipid Rafts: Pr55gag Associates with Membrane Domains That Are Largely Resistant to Brij98 but Sensitive to Triton X-100

Kirsi Holm, Katarzyna Weclewicz,{dagger} Roger Hewson,{ddagger} and Maarit Suomalainen*

Department of Biosciences at Novum, Karolinska Institutet, S-141 57 Huddinge, Sweden

Received 16 September 2002/ Accepted 25 January 2003

The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55gag. We have analyzed whether Pr55gag has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55gag has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55gag particles (VLPs) yield buoyant Pr55gag complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55gag complexes cannot be taken as a proof for raft association of Pr55gag, since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55gag. However, Pr55gag might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55gag. Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55gag complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55gag VLPs.

* Corresponding author. Mailing address: Department of Biosciences at Novum, Karolinska Institutet, S-141 57 Huddinge, Sweden. Phone: 46-8-608 9133. Fax: 46-8-774 5538. E-mail: maarit.suomalainen{at}cbt.ki.se.

{dagger} Present address: Clinic of Neurology, University Hospital Huddinge, Karolinska Institutet, S-141 57 Huddinge, Sweden.

{ddagger} Present address: Special Pathogens/Virology, Centre for Applied Microbiology and Research, Porton Down, Salisbury SP4 0JG, United Kingdom.

Journal of Virology, April 2003, p. 4805-4817, Vol. 77, No. 8
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.8.4805-4817.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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