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

Abstract

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.

Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120

“…This dual effect appears to be analogous to the conformational switch region that has been recently described for the fusion protein (F) of a paramyxovirus (34), in which a region near the C-HR regulates both activation of F-mediated fusion and 6HB stability. Residues in the N-HR region of gp41 have previously been reported to be important for contacts with gp120 (6,16,25), further supporting the idea that this residue may be involved in a conformational switch. Notably, residue 577 lies in a hydrophobic pocket region of the N-HR, which has been proposed to be an important region for stabilizing the coiled coil (13) and a good target for developing inhibitors (7).…”

Conserved Residue Lys574 in the Cavity of HIV-1 Gp41 Coiled-coil Domain Is Critical for Six-helix Bundle Stability and Virus Entry

“…The helical packing interactions in the HIV-1 gp41 core have been extensively explored by mutagenesis and it was shown that a number of mutations were able to destabilize the 6-HB and abolish viral infectivity (14,37,38). With mutational analysis, we have investigated the role of the residue Lys 574 located in the NHR cavity.…”

“…Here we show, for the first time, that residue Lys 574 , which is located at the heptad b position, determines the stability and conformation of the gp41 core structure. Furthermore, it is believed that the structure of the HIV-1 gp120-gp41 complex exists as a trimer and that the highly conserved 4-3 hydrophobic repeat in the N-terminal portion of gp41 plays a critical role in oligomer formation and stability (14,40). Single substitutions of isoleucine or leucine with proline within the NHR abolished Envmediated membrane fusion activity, but did not interfere with Env oligomerization (41,42), suggesting that the putative coiled-coil domain is required for virus entry.…”

Potent HIV fusion inhibitors against Enfuvirtide-resistant HIV-1 strains

“…1B) that has a tendency to bind lipid membranes (20). The helical packing interactions between the NHR and CHR play an essential role in viral infectivity (21)(22)(23).…”