Originally published In Press as doi:10.1074/jbc.M301627200 on March 24, 2003
J. Biol. Chem., Vol. 278, Issue 22, 20278-20285, May 30, 2003
Covalent Trimers of the Internal N-terminal Trimeric Coiled-coil of gp41 and Antibodies Directed against Them Are Potent Inhibitors of HIV Envelope-mediated Cell Fusion*
John M. Louis,
Issa Nesheiwat,
LengChee Chang,
G. Marius Clore 
and
Carole A. Bewley 
From the
Laboratories of Chemical Physics and Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
We have engineered two soluble, covalently linked, trimeric polypeptides, N35CCG-N13 and N34CCG comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit human immunodeficiency virus, type 1 (HIV-1) envelope (Env)-mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the prehairpin intermediate state. The IC50 values for N35CCG-N13 and N34CCG are 
15 and 95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env-mediated cell fusion using Env from the T cell tropic strain LAV. Polyclonal antibodies were raised against N35CCG-N13 and a tightly binding fraction of anti-N35CCG-N13 inhibits T cell and macrophage tropic HIV-1 Env-mediated cell fusion with respective IC50 values of 0.5 and 1.5 µg/ml at 37 °C. The tightly binding anti-N35CCG-N13 antibody fraction targets the exposed internal trimeric coiled-coil in the prehairpin intermediate state of gp41 in a manner analogous to peptides derived from the C region of the gp41 ectodomain. The potency of the tightly binding anti-N35CCG-N13 antibody fraction in the fusion assay is comparable with that of the broadly neutralizing monoclonal antibody 2G12. These results indicate that N35CCG-N13 is a potential anti-HIV therapeutic agent and represents a suitable immunogen for the generation of neutralizing monoclonal antibodies targeted to the internal trimeric coiled-coil of gp41. The data on the tightly binding anti-N35CCG-N13 antibody fraction demonstrate that the internal trimeric coiled-coil of gp41 in the prehairpin intermediate state is accessible to antibodies and that access is not restricted by either antibody size or the presence of a kinetic barrier.
Received for publication, February 14, 2003
, and in revised form, March 24, 2003.
* This work was supported by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C. and C. A. B.). 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.
To whom correspondence may be addressed: Laboratory of Chemical Physics, Bldg. 5, Rm. B1–30I, NIDDK, National Institutes of Health, Bethesda, MD 20892-0510. Tel.: 301-496-0782; Fax: 301-496-0825; E-mail: mariusc{at}intra.niddk.nih.gov. To whom correspondence may be addressed: Laboratory of Bioorganic Chemistry, Bldg. 8, Rm. 1A-02, NIDDK, National Institutes of Health, Bethesda, MD 20892-0820. Tel.: 301-594-5187; E-mail: cb194k{at}nih.gov.
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
