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Journal of Virology, January 2001, p. 834-843, Vol. 75, No. 2
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.2.834-843.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Design and Use of an Inducibly Activated Human Immunodeficiency Virus Type 1 Nef To Study Immune Modulation

Scott F. Walk, Melissa Alexander, Bernhard Maier, Marie-Louise Hammarskjold, David M. Rekosh, and Kodi S. Ravichandran*

Carter Immunology Center, Myles H. Thaler Center for AIDS and Human Retrovirus Research and the Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908

Received 30 June 2000/Accepted 25 October 2000

The Nef protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to enhance the infectivity of virus particles, downmodulate cell surface proteins, and associate with many intracellular proteins that are thought to facilitate HIV infection. One of the challenges in defining the molecular events regulated by Nef has been obtaining good expression of Nef protein in T cells. This has been attributed to effects of Nef on cell proliferation and apoptosis. We have designed a Nef protein that is readily expressed in T-cell lines and whose function is inducibly activated. It is composed of a fusion between full-length Nef and the estrogen receptor hormone-binding domain (Nef-ER). The Nef-ER is kept in an inactive state due to steric hindrance, and addition of the membrane-permeable drug 4-hydroxytamoxifen (4-HT), which binds to the ER domain, leads to inducible activation of Nef-ER within cells. We demonstrate that Nef-ER inducibly associates with the 62-kDa Ser/Thr kinase and is localized to specific membrane microdomains (lipid rafts) only after activation. Using this inducible Nef, we also compared the specific requirements for CD4 and HLA-A2 downmodulation in a SupT1 T-cell line. Half-maximal downmodulation of cell surface CD4 required very little active Nef-ER and occurred as early as 4 h after addition of 4-HT. In contrast, 50% downmodulation of HLA-A2 by Nef required 16 to 24 h and about 50- to 100-fold-greater concentrations of 4-HT. These data suggest that HLA-A2 downmodulation may require certain threshold levels of active Nef. The differential timing of CD4 and HLA-A2 downmodulation may have implications for HIV pathogenesis and immune evasion.

* Corresponding author. Mailing address: Carter Immunology Center, University of Virginia, Bldg. MR4, Rm. 4012F, P.O. Box 801386, HSC, Charlottesville, VA 22908-1386. Phone: (804) 243-6093. Fax: (804) 924-1221. E-mail: Ravi{at}virginia.edu.

Journal of Virology, January 2001, p. 834-843, Vol. 75, No. 2
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.2.834-843.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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