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Journal of Virology, November 2001, p. 11146-11156, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.11146-11156.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Structural and Functional Analysis of Interhelical Interactions in the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein by Alanine-Scanning Mutagenesis

Min Lu,^1,* Marisa O. Stoller,² Shilong Wang,¹ Jie Liu,¹ Melinda B. Fagan,^2, and Jack H. Nunberg^2,*

Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021,¹ and Montana Biotechnology Center, The University of Montana, Missoula, Montana 59812²

Received 23 May 2001/Accepted 8 August 2001

Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.

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^* Corresponding author. Mailing address for Jack H. Nunberg: Montana Biotechnology Center, The University of Montana, Missoula, MT 59812. Phone: (406) 243-6421. Fax: (406) 243-6425. E-mail: nunberg{at}selway.umt.edu. Mailing address for Min Lu: Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021. Phone: (212) 746-6562. Fax: (212) 746-8875. E-mail: mlu{at}med.cornell.edu.

Present address: Department of Philosophy, University of Texas, Austin, TX 78712.

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Journal of Virology, November 2001, p. 11146-11156, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.11146-11156.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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