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J. Biol. Chem., Vol. 280, Issue 24, 23138-23146, June 17, 2005

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Design, Expression, and Immunogenicity of a Soluble HIV Trimeric Envelope Fragment Adopting a Prefusion gp41 Configuration^*

Zhi-Song Qiao, Mikyung Kim, Bruce Reinhold, David Montefiori¶, Jia-huai Wang||**, and Ellis L. Reinherz

From the Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute, the Departments of Medicine, ^||Pediatrics, and ^**Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and the ^¶Department of Surgery, Duke University, Medical Center, Durham, North Carolina 27710

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys⁶⁶⁵ that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.

Received for publication, December 23, 2004 , and in revised form, April 12, 2005.

^* This work was supported by National Institutes of Health Grant AI43649. 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 should be addressed: Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3412; Fax: 617-632-3351; E-mail: ellis_reinherz{at}dfci.harvard.edu.

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