Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer

<div><p>Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI.</p></div

Table of Heterotrimers

<p>Table of Heterotrimers</p

Structures of the wild type and mutant gp41 trimers-of-hairpins.

<p>(<b>A</b>) Helical wheel diagram of the gp41 trimer-of-hairpins with residue positions labeled <i>a</i> through <i>g</i> based on the 3,4-hydrophobic heptad repeat. The relative positions of Val₅₄₉ (green) and Gln₅₅₂ (red) within the N-HR coiled coil are designated. (<b>B</b>) Ribbon diagram of the wild type gp41 trimer-of-hairpins formed by peptides N36 (gray) and C34 (blue) based on the Env_HXB2 sequence (PDB ID: 1AIK. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006098#ppat.1006098.ref012" target="_blank">12</a>]). The side chains of Val₅₄₉ (green) and Gln₅₅₂ (red) are depicted in stick representation. (<b>C</b>) Ribbon diagram of a gp41 trimer-of-hairpins containing the Q552R substitution. The side chains of Arg₅₅₂ are shown in stick representation (red) while the unwound region of the distorted N40 segment is colored orange. (<b>D</b>) Ribbon diagram of the gp41 trimer-of-hairpins containing the V549E substitution. The side chains of Glu₅₄₉ are shown in stick representation (green). The structures in <b>C</b> and <b>D</b> were obtained using a gp41 construct (NC1) containing 40 amino acids of the N-HR segment (gray) and 37 amino acids of the C-HR segment (blue).</p

Probing exposure of the gp41 N-HR coiled coil.

<p>(<b>A</b>) T20 sensitivity of Ww (black), Wm (red), Mw (green) and Mm (blue) viruses where mutant Env protomers contained the L544S substitution. Viral samples were produced from cells expressing equal levels of Env A and Env B protomers. (<b>B</b>) IC₅₀ values for T20 (dark gray) and di-C37 (light gray) against HIV-1 produced from cells expressing equal levels of Env A and Env B protomers. T20 is affinity-dependent while di-C37 is kinetically restricted. (<b>C</b> and <b>D</b>) IC₅₀ values for T20 (<b>C</b>) and di-C37 (<b>D</b>) against HIV-1 produced from cells expressing Env A and Env B protomers at the indicated ratios. IC₅₀ values (<b>B</b>, <b>C</b> and <b>D</b>) were obtained from fits of inhibitor titration data to the Langmuir equation (solid lines in <b>A</b>). (<b>E-H</b>) As described for panels <b>A-D</b> except that the mutant protomers contained the V549A substitution and C37-KYI was the kinetically restricted inhibitor. Data points represent the means ± SEM from three or more independent experiments. The target cells were U87.CD4.CXCR4.</p

Structural transformations of the HIV-1 Env trimer during viral entry.

<p>(<b>A</b>) In the native, prefusogenic conformation (PDB ID: 5FYL, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006098#ppat.1006098.ref011" target="_blank">11</a>]), gp41 subunits (ribbon diagram) are held in a metastable conformation by a canopy of gp120 subunits (green space-filling model). In the orientation shown, the viral membrane is at the base of the modeled structure. Cellular receptor binding to gp120 releases constraints on the gp41 N-HR and C-HR segments, enabling the N-HR coiled coil to form and extend the gp41 N-termini toward the target cell membrane. The disposition of gp120 subunits in this transient intermediate state is unknown. Ultimately, the N-HR and C-HR segments collapse into a six-helix bundle (PDB ID: 1AIK, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006098#ppat.1006098.ref012" target="_blank">12</a>]) that stabilizes the postfusogenic gp41 trimer-of-hairpins. In this conformation, the gp41 N-terminal fusion peptide and transmembrane segments (not depicted), as well as their associated membranes, are on the same side of the molecule (top). The fusion inhibitors used in this study target the N-HR coiled coil and C-HR segments transiently exposed during the prehairpin intermediate state. (<b>B</b>) Closeup of a single gp41 ectodomain (ribbon diagram) in its metastable native conformation cradled by its cognate gp120 subunit (green). From N- to C-terminus, the regions of the gp41 ectodomain are color coded as follows: fusion peptide and fusion-peptide proximal region—red; N-terminal heptad repeat (N-HR)—grey; disulfide-bonded loop—orange; C-terminal heptad repeat (C-HR)—blue. The membrane proximal-external region (MPER) is not shown. (<b>C</b>) Schematic of a single Env protomer highlighting gp120 and gp41 segments modified for this study. Fusion inhibitor binding sites in the N-HR and C-HR regions and the relative positions of escape mutations are indicated. The C37 binding site is targeted by both C37-KYI and di-C37, while the 5-Helix binding site is targeted by both 5H_WT and 5H_LAVA.</p

Hypothetical model of the early structural changes in Env leading to exposure of the N-HR coiled coil.

<p>(<b>A</b>) Proposed transitions for an Env trimer that encounters three CD4 molecules. In the native state, gp120 subunits (green) constrain the N-HR segments (grey) in a metastable conformation in which the coiled coil is only partially formed (schematically represented as ovals). The binding of the first CD4 (orange) alters trimer structure enabling formation of the full N-HR coiled coil (schematically represented as circles with <i>e</i>- and <i>g</i>-positions of the heptad repeat designated). The CD4-bound gp120 subunit disengages but only partially exposes C-peptide binding sites on either side of its cognate N-HR helix. The remaining two gp120 subunits remain closely associated with the coiled coil, thereby blocking C-peptide access to all three binding sites. The second CD4-gp120 interaction causes this gp120 subunit to disengage, opening up a single C-peptide binding site on the N-HR coiled coil (designated by the red arrow). The other two C-peptide binding sites remain partially occluded by the one gp120 that remains attached. Upon association of the third CD4, the last gp120 disengages and the N-HR coiled coil with its three C-peptide binding sites becomes fully exposed. Ultimately, these sites become occupied by gp41 C-HR regions (blue) as Env transitions into its fusogenic trimer-of-hairpins conformation. The model describes the putative transitions of homotrimeric Envs, including the Q552R/N637K variant of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006098#ppat.1006098.g007" target="_blank">Fig 7</a>. (<b>B</b> and <b>C</b>) Proposed transitions for an Env trimer that encounters only two (<b>B</b>) or one (<b>C</b>) CD4 molecule. The gp120 subunits that do not interact with CD4 are colored brown. The asterisk indicates a spontaneous, CD4-independent conformational change. The models describe the putative transitions of A₂B (<b>B</b>) and AB₂ (<b>C</b>) heterotrimers.</p

Probing exposure of the three gp41 C-HR domains.

<p>(<b>A</b>) 5H_LAVA sensitivity of Ww (black), Wm (red), Mw (green) and Mm (blue) viruses where mutant Env protomers contained the N656D substitution. Viral samples were produced from cells expressing equal levels of Env A and Env B protomers. (<b>B</b>) IC₅₀ values for 5H_LAVA (dark gray) and 5H_WT (light gray) against HIV-1 produced from cells expressing equal levels of Env A and Env B protomers. 5H_LAVA is affinity-dependent while 5H_WT is kinetically restricted. (<b>C</b> and <b>D</b>) IC₅₀ values for 5H_LAVA (<b>C</b>) and 5H_WT (<b>D</b>) against HIV-1 produced from cells expressing Env A and Env B protomers at the indicated ratios. IC₅₀ values (<b>B</b>, <b>C</b> and <b>D</b>) were obtained as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006098#ppat.1006098.g004" target="_blank">Fig 4</a>. Data points represent the means ± SEM from five independent experiments. The target cells were U87.CD4.CXCR4.</p

Combining functional complementation with fusion inhibitor escape mutations to assess gp41 exposure.

<p>HIV-1 was collected from cells co-expressing Env A and Env B protomers containing either wild type or mutant gp41 subunits. Viral populations are labeled with a two-letter nomenclature as follows: the first letter (upper case) indicates the gp41 status (wild type—W, mutant—M) of the CD4-interacting Env A protomer; the second letter (lower case) indicates the gp41 status (wild type—w, mutant—m) of the coreceptor-binding Env B protomer. The fusogenic A₂B and AB₂ heterotrimers for each viral population are schematically depicted with mutant gp41 subunits colored red. The number of high affinity fusion inhibitor binding sites per trimer is indicated in the small box at the lower right. Nonfusogenic Env homotrimers resulting from coexpression of Env A and Env B are not portrayed.</p