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J. Biol. Chem., Vol. 282, Issue 11, 8207-8218, March 16, 2007

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Identifying the Membrane Proteome of HIV-1 Latently Infected Cells^*

Reem Berro, Cynthia de la Fuente¹, Zachary Klase^¶, Kylene Kehn², Lida Parvin^||, Anne Pumfery³, Emmanuel Agbottah, Akos Vertes^||, Sergei Nekhai^**, and Fatah Kashanchi⁴

From the Genetics Program, Department of Biochemistry and Molecular Biology, ^¶Department of Immunology, and ^||Department of Chemistry, The George Washington University, School of Medicine, Washington, D. C. 20037, ^**Department of Biochemistry/Center for Sickle Cell Disease, Howard University, Washington, D. C. 20059, and The Institute for Genomic Research, Rockville, Maryland 20850

Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.

Received for publication, July 3, 2006 , and in revised form, January 18, 2007.

^* This work was supported by grants from the George Washington University REF funds (to R. B., F. K., and A. V.), a Snyder award, and National Institutes of Health Grants AI065236 and AI043894 (to F. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Current address: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10021.

² Current address: Counterterrorism and Forensic Science Research Unit, Federal Bureau of Investigation Laboratory, Quantico, VA 22135.

³ Current address: Dept. of Biology, Seton Hall University, South Orange, NJ 07079.

⁴ To whom correspondence should be addressed: The George Washington University, 2300 I St., NW, Ross Hall, Rm. 551, Washington, D. C. 20037. Tel.: 202-994-1781; Fax: 202-994-1780; E-mail: bcmfxk{at}gwumc.edu.

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