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Journal of Virology, September 2006, p. 8510-8520, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00420-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

Marina Kovaleva ,1,{dagger},{ddagger} Ingo Bussmeyer,1,{dagger} Björn Rabe,1 Joachim Grötzinger,1 Enge Sudarman,2 Jutta Eichler,2 Udo Conrad,3 Stefan Rose-John,1* and Jürgen Scheller1

Department of Biochemistry, Christian Albrechts Universität, Kiel, Germany,1 Gesellschaft für Biotechnologische Forschung GmbH, Braunschweig, Germany,2 Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben (IPK), Gatersleben, Germany3

Received 28 February 2006/ Accepted 7 June 2006

Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.

* Corresponding author. Mailing address: Department of Biochemistry, Christian Albrechts Universität zu Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany. Phone: 49-431-880 3336. Fax: 49-431-880 5007. E-mail: rosejohn{at}biochem.uni-kiel.de.

{dagger} M.K. and I.B. contributed equally to this work.

{ddagger} Present address: North-West Cancer Research Fund Institute, University of Wales, Bangor, United Kingdom.

Journal of Virology, September 2006, p. 8510-8520, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00420-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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