Structural and Functional Properties of the Membranotropic HIV-1 Glycoprotein gp41 Loop Region Are Modulated by Its Intrinsic Hydrophobic Core

Jiayin Qiu,Avraham Ashkenazi,Shuwen Liu,Yechiel Shai

doi:10.1074/jbc.m113.496646

Abstract

The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well.

Highlights

human immunodeficiency virus type 1 (HIV-1) infectivity is decreased by specific mutations that alter the hydrophobicity level in the HIV-1 gp41 loop core
Bioinformatics Analysis of Loop Core Sequences in the Fusion Proteins of HIV and SIV—We observed an unusual Lys incorporated in the hydrophobic core of the HIV-1 gp41 loop region (Lys-601) that disrupts the hydrophobic continuity of the core
Emerging studies suggest that the loop region and its conserved cysteines participate in the actual lipid mixing process thereby stabilizing the gp41 hairpin conformation [17, 20, 21]

Summary

Background

HIV-1 infectivity is decreased by specific mutations that alter the hydrophobicity level in the HIV-1 gp loop core. We utilized biochemical and biophysical approaches to study the affect of the K601A and L602A mutations on the function and structure of the loop region inside and outside the membrane. For this purpose, peptides corresponding to the loop region were prepared with the mutations K601A and L602A that have an opposite effect on the hydrophobic level of the core (Fig. 1D). The L602A mutation, but not that of the K601A, decreased the lipid mixing ability of the loop These defects were in correlation with alterations in the structure and proper conformation of the loop as revealed by antibody binding and circular dichroism spectroscopy. The results are discussed in the context of the HIV-1 gp41-mediated membrane fusion event

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION