The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/1998/11/1917/ $5.00
The Journal of Experimental Medicine, Volume 188, Number 10, November 16, 1998 1917-1927

Articles

A New Monoclonal Antibody, mAb 4A12, Identifies a Role for the Glycosaminoglycan (GAG) Binding Domain of RANTES in the Antiviral Effect against HIV-1 and Intracellular Ca2+ Signaling

Jennifer M. Burns, Robert C. Gallo, Anthony L. DeVico, and George K. Lewis

From the Divisions of Basic Science and Vaccine Research, Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201; and the Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201

The β-chemokine RANTES (regulated on activation, normal T cell expressed and secreted) suppresses the infection of susceptible host cells by macrophage tropic strains of HIV-1. This effect is attributed to interactions of this chemokine with a 7-transmembrane domain receptor, CCR5, that is required for virus–cell fusion and entry. Here we identify domains of RANTES that contribute to its biological activities through structure–function studies using a new monoclonal antibody, mAb 4A12, isolated from mice immunized with recombinant human RANTES. This monoclonal antibody (mAb) blocked the antiviral activity of RANTES in infectivity assays with HIV-1Bal, and inhibited the mobilization of intracellular Ca2+ elicited by RANTES, yet recognized this chemokine bound to cell surfaces. Epitope mapping using limited proteolysis, reversed phase high-performance liquid chromatography, and mass spectrometry suggest that residues 55–66 of RANTES, which include the COOH-terminal {alpha}-helical region implicated as the glycosaminoglycan (GAG) binding domain, overlap the determinant recognized by mAb 4A12. This is supported by affinity chromatography studies, which showed that RANTES could be eluted specifically by heparin from a mAb 4A12 immunoaffinity matrix. Removal of cell surface GAGs by enzymatic digestion greatly reduced the ability of mAb 4A12 to detect RANTES passively bound on cell surfaces and abrogated the ability of RANTES to elicit an intracellular Ca2+ signal. Taken together, these studies demonstrate that the COOH-terminal {alpha}-helical region of RANTES plays a key role in GAG-binding, antiviral activity, and intracellular Ca2+ signaling and support a model in which GAGs play a key role in the biological activities of this chemokine.

Key Words: β-chemokines • human immunodeficiency virus 1 • monoclonal antibody • signaling • antiviral effect

Address correspondence to Anthony L. DeVico or George K. Lewis, Institute of Human Virology, 725 W. Lombard St., Baltimore, MD 21201. Phone: 410-706-4688; Fax: 410-706-4695; E-mail: devico @umbi.umd.edu or lewisg{at}umbi.umd.edu

The skillful technical assistance of Mr. Robert Tuskan and Ms. Beverly Lamb is genuinely appreciated. We thank Dr. Roberta Kamin-Lewis for critical comments. We thank Dr. K. Swiderek, Beckman Research Institute of the City of Hope, City of Hope, CA for sequence analysis.

A.L. DeVico and G.K. Lewis contributed equally to this work.

This work was submitted by J.M. Burns in partial fulfillment of the requirements of a Ph.D. thesis in the Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201.

Abbreviations used: FBS, fetal bovine serum; GAG, glycosaminoglycan; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T cell expressed and secreted; TCID50, tissue culture dose that infects 50% of cultures.


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