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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.
Nunberg2,*
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.
*
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.
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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