Structural and functional analysis of interhelical interactions in the human immunodeficiency virus type 1 gp41 envelope glycoprotein by alanine-scanning mutagenesis.
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.
Lu
M
M
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA. mlu@med.cornell.edu
Stoller
M O
MO
Wang
S
S
Liu
J
J
Fagan
M B
MB
Nunberg
J H
JH
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002459:Cell Fusion; D003460:Crystallization; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000502:physiology; D015497:HIV-1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation
D015700:HIV Envelope Protein gp41; D000409:Alanine