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Journal of Virology, September 2006, p. 8796-8806, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.02159-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mobility of Human Immunodeficiency Virus Type 1 Pr55Gag in Living Cells{dagger}

Candace Y. Gomez1,2 and Thomas J. Hope1*

Department of Cell & Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611,1 Department of Microbiology and Immunology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 606122

Received 13 October 2005/ Accepted 15 June 2006

Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

* Corresponding author. Mailing address: Department of Cell & Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611. Phone: (312) 503-1360. Fax: (312) 503-2696. E-mail: thope{at}northwestern.edu.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, September 2006, p. 8796-8806, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.02159-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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