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Journal of Virology, May 2000, p. 4746-4754, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Modifications That Stabilize Human Immunodeficiency Virus Envelope Glycoprotein Trimers in Solution

Xinzhen Yang,1,2 Lauren Florin,1 Michael Farzan,1,2 Peter Kolchinsky,1 Peter D. Kwong,3 Joseph Sodroski,1,2,4 and Richard Wyatt1,5,*

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute,1 Department of Pathology2 and Department of Medicine,5 Harvard Medical School, and Department of Immunology and Infectious Diseases, Harvard School of Public Health,4 Boston, Massachusetts 02115, and Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 100323

Received 17 November 1999/Accepted 21 February 2000

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.

* Corresponding author. Mailing address: Dana-Farber Cancer Institute, 44 Binney St., JFB 815, Boston, MA 02115. Phone: (617) 632-3905. Fax: (617) 632-3113. E-mail: Richard_Wyatt{at}dfci.harvard.edu.

Journal of Virology, May 2000, p. 4746-4754, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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