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Journal of Virology, November 2007, p. 12596-12607, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01088-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Sequence of Human Immunodeficiency Virus Type 1 (HIV-1) Gag Localization and Oligomerization Monitored with Live Confocal Imaging of a Replication-Competent, Fluorescently Tagged HIV-1{triangledown} ,{dagger}

Wolfgang Hübner,1 Ping Chen,1 Armando Del Portillo,1 Yuxin Liu,1 Ronald E. Gordon,2 and Benjamin K. Chen1*

Department of Medicine, Division of Infectious Disease, Immunology Institute, Mount Sinai School of Medicine, New York, New York 10029,1 Department of Pathology, Mount Sinai School of Medicine, New York, New York 100292

Received 21 May 2007/ Accepted 21 August 2007

The assembly of infectious human immunodeficiency virus (HIV) requires that Gag transport and oligomerization be coordinated with its association with other viral proteins, viral RNAs, and cellular membranes. We have developed a replication-competent HIV type 1 molecular clone that carries a Gag-internal or interdomain green fluorescent protein (iGFP) fusion to reveal a physiologically accurate temporal sequence of Gag localization and oligomerization during the formation of infectious HIV. This recombinant HIV is as infectious as native HIV in single-round infectivity assays, validating its use for trafficking studies. It replicates robustly in permissive MT4 cells and is infectious, yet it spreads poorly in other T-cell lines. Immunofluorescence of Gag-iGFP showed a pattern very similar to that of native Gag. However, the intense plasma membrane Gag-iGFP fluorescence contrasts markedly with its immunofluorescence at this site, indicating that many Gag epitopes can be masked by oligomerization. Consistent with this, fluorescence resonance energy transfer studies visualized intense Gag oligomerization at the plasma membrane and weaker oligomerization at cytoplasmic sites. Four-dimensional, time-lapse confocal imaging reveals a temporal progression of Gag distribution over hours in which Gag is initially diffusely localized within the cytoplasm. Plasma membrane signals then accumulate as Gag levels increase and vesicular association appears late, only after plasma membrane site signals have reached high intensity. Lastly, the cell rounds up and HIV protease activation induces diffuse fluorescence throughout the cell. These distinct phases reveal a natural progression of Gag trafficking during the viral gene expression program. HIV Gag-iGFP is a useful tool for dissecting mechanisms of viral assembly and transmission.

* Corresponding author. Mailing address: One Gustave Levy Place, Box 1630, New York, NY 10029. Phone: (212) 659-9408. Fax: (212) 849-2525. E-mail: ben.chen{at}mssm.edu

{triangledown} Published ahead of print on 29 August 2007.

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

Journal of Virology, November 2007, p. 12596-12607, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01088-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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