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. 2008 Feb 22;376(3):786-97.
doi: 10.1016/j.jmb.2007.12.001. Epub 2007 Dec 7.

Peptide mimic of the HIV envelope gp120-gp41 interface

Sunghwan Kim  1 , Hong-Bo Pang, Michael S Kay
Affiliations

Peptide mimic of the HIV envelope gp120-gp41 interface

Sunghwan Kim et al. J Mol Biol. .

Abstract

The human immunodeficiency virus envelope glycoprotein (Env) is composed of surface (gp120) and transmembrane (gp41) subunits, which are noncovalently associated on the viral surface. Human immunodeficiency virus Env mediates viral entry after undergoing a complex series of conformational changes induced by interaction with cellular CD4 and a chemokine coreceptor. These changes propagate from gp120 to gp41 via the gp120-gp41 interface, ultimately exposing gp41 and allowing it to form the trimer-of-hairpins structure that provides the driving force for membrane fusion. Key unresolved questions about the gp120-gp41 interface include the specific regions of gp41 and gp120 involved, the mechanism by which receptor and coreceptor-binding-induced conformational changes in gp120 are communicated to gp41, how trimer-of-hairpins formation is prevented in the prefusogenic gp120-gp41 complex, and, ultimately, the structure of the prefusion gp120-gp41 complex. Here, we develop a biochemical model system that mimics a key portion of the gp120-gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and C-peptide region binds primarily to the gp120 C5 region and that this interaction is incompatible with trimer-of-hairpins formation. Based on these data, we propose that in prefusogenic Env, gp120 sequesters the gp41 C-peptide region away from the N-trimer region, preventing trimer-of-hairpins formation until coreceptor binding disrupts this interface. This model system is a valuable tool for studying the gp120-gp41 complex, conformational changes induced by CD4 and coreceptor binding, and the mechanism of membrane fusion.

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Figures

Figure. 1
Figure. 1. HIV entry model and schematic of gp41 and gp120 fragments
(A) Working model of HIV entry. (B) Schematic of gp41 fragments. gp41 consists of the ectodomain (ED), transmembrane domain (TM), and cytoplasmic tail domain (CT). Constructs N-DSL49 (540-666), DSL49 (584-666), DSL20 (584-637), C49 (618-666), C43 (624-666), C20 (618-637), and DSL (584-622) are shown. DSL has one intramolecular disulfide bond, indicated by the dashed line between Cys598 and 604. All gp41 fragments contain a C-terminal His-tag. (C) JRFL-gp120 deletion constructs. ΔC1 (33-82), ΔC5 (493-511), ΔV1/V2 (128-194), and ΔV3 (303-323) are indicated. For ΔC1, ΔV1/V2 and ΔV3, the deleted loops are substituted with GS, GAG and GA, respectively. Core gp120 contains all of the deletions (ΔV1/V2/V3 and ΔC1/C5). Amino acid numbering is based on the prototypic HXB2 gp160 sequence.
Figure. 2
Figure. 2. Binding of gp41 fragments to gp120
(A) gp41 fragments were incubated with gp120 in the presence or absence of untagged sCD4 and precipitated with Ni++ beads. Eluents were deglycosylated and analyzed by non-reducing SDS-PAGE Western blot with anti-gp120 antibody. Mock lane contains gp120 without any gp41 fragment. (B) Quantification of monomeric gp120 binding from panel A. Binding percentage was normalized to the amount of gp120 amount precipitated by CD4-H6 in the absence of untagged sCD4. (C) DSL49, DSL20, and C49 incubated with gp120 in the presence or absence of sCD4 and 5-helix as indicated. (D) Schematic diagram of 5-helix binding to gp41 fragments.
Figure. 3
Figure. 3. Interaction between gp41 fragments and gp120 deletion mutants or MBP-C1/C5
(A) gp120 deletion constructs were co-precipitated with DSL49 and DSL20 ± sCD4 as described in Fig. 2A. (B) Quantification of monomeric gp120 binding from panel A, normalized as in Fig. 2B. (C) Co-precipitation of 10 μM MBP or MBP-C1/C5 with 2 μM DSL49. As a negative control, His-tagged ubiquitin (Ub-H6) was used. Loading control contains 1 μM MBP and MBP-C1/C5 (corresponding to 50% of maximal binding to 2 μM DSL49) and 2 μM DSL49 and Ub-H6 (100% of maximal binding). Precipitated proteins by Ni++ beads were analyzed by SDS-PAGE.
Figure. 4
Figure. 4. Binding of DSL20 mutants to gp120 and cell surface
(A) DSL20 mutant constructs: Δ13-DSL20 (597-637), L20 (605-637) and DSL20ss (C598S and C604S). (B) DSL49, DSL20, and DSL20 mutants were incubated with gp120 in the presence of sCD4. Mock lane contains only gp120 and sCD4. (C) Binding of DSL49, DSL49ss, DSL20, and DSL20ss to HOS cells. Right panel shows the effect of 5-helix addition on cell surface binding. Bound proteins on the cell surface were analyzed by Western blot with anti-His tag antibody. Mock lanes were prepared without addition of gp41 fragment. (D) Binding of DSL49, DSL49ss, DSL49-NEM, DSL49-Ac, and DSL49-red to HOS cells.
Figure 5
Figure 5. Immunostaining of DSL49 and its DSL49ss on cellular membrane
DSL49 or DSL49ss was incubated with HOS-pBABE cells and stained with rabbit anti-His6 antibody and Alexa Fluor 568 goat anti-rabbit antibody. Cells were placed on the slide and photographed at 40X and 100X magnification.
Figure 6
Figure 6. CD4-induced conformational changes in gp120
Structural comparison between unliganded (left) and CD4-bound gp120 (right). Structures are rendered from PDB accession numbers 2BF1 and 1RZJ, respectively ,, using PyMol. Blue and red spheres indicate N-terminus and C-terminus, respectively. Yellow and green indicate V1/V2 stem and V3 stem, respectively.
Figure 7
Figure 7. Revised model of HIV entry
Based on the results of this study, we propose this altered model of HIV entry. CD4 binding induces formation of the pre-hairpin intermediate, in which the N-trimer region is exposed and the fusion peptide embedded in the target cell membrane. In this intermediate, the DSL and C-peptide regions of gp41 interact with gp120, preventing association with the N-trimer region. Coreceptor binding to gp120 triggers conformational changes that weaken the gp120/gp41 interface. The liberated C-peptide region can then interact with the N-trimer region to form the trimer of hairpins structure, leading to membrane fusion and viral entry.
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