Published online before print October 4, 2005

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0105043

Received for publication January 24, 2005.
Revised July 26, 2005.
Accepted for publication July 29, 2005.

Article

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association

Marina Zaitseva ^*@, Tatiana Romantseva ^*, Jody Manischewitz ^*, Jiun Wang , David Goucher , and Hana Golding ^*

Divisions of *Viral Products, Center for Biologics Evaluation and Research, Monoclonal Antibodies, and Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

^@ To whom correspondence should be addressed. E-mail: zaitseva{at}cber.fda.gov.

Abstract

Activation of peripheral CD4⁺ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63⁺ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.

Key Words: chemokine receptors • cell activation

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