Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure

The membrane proximal external region (MPER) of HIV-1 glycoprotein (gp) 41 is involved in viral-host cell membrane fusion. It contains short amino acid sequences that are binding sites for the HIV-1 broadly neutralizing antibodies 2F5, 4E10, and 10E8, making these binding sites important targets for HIV-1 vaccine development. We report a high-resolution structure of a designed MPER trimer assembled on a detergent micelle. The NMR solution structure of this trimeric domain, designated gp41-M-MAT, shows that the three MPER peptides each adopt symmetric α-helical conformations exposing the amino acid side chains of the antibody binding sites. The helices are closely associated at their N termini, bend between the 2F5 and 4E10 epitopes, and gradually separate toward the C termini, where they associate with the membrane. The mAbs 2F5 and 4E10 bind gp41-M-MAT with nanomolar affinities, consistent with the substantial exposure of their respective epitopes in the trimer structure. The traditional structure determination of gp41-M-MAT using the Xplor-NIH protocol was validated by independently determining the structure using the DISCO sparse-data protocol, which exploits geometric arrangement algorithms that guarantee to compute all structures and assignments that satisfy the data.

Section: Discussionmentioning

confidence: 71%

Structure of an HIV-1–neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer

Reardon¹,

Sage²,

Dennison

et al. 2014

“…Solubilized Envs, either in their native form or in their activated intermediate form (IAS), were isolated by sedimentation in a linear 5-20% sucrose gradient in HNC buffer containing 0.05% Triton X-100, superimposed by a 0-0.2% glutaraldehyde gradient (30). Trimers of disulfidelinked SU-TM complexes were identified in gradient fractions by BN/PAGE and silver staining and used for EM analyses.…”

Section: Pagementioning

confidence: 99%

“…Isolated Envs were adsorbed to grids coated with a holey carbon film, blotted, and plunge-frozen in ethane using an FEI Vitrobot. Micrographs were recorded with a JEOL 2100F transmission electron microscope at 200 kV as described (30). In brief, particles were selected and visually verified in boxer, and corrected for the contrast transfer function using ctfit, both in EMAN 1 (http://blake.bcm.edu/emanwiki/EMAN).…”

Section: Pagementioning

confidence: 99%

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

Löving

Wu²,

Sjöberg

et al. 2012

Self Cite

The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.three-dimensional structure | retrovirus | spike protein T he spike protein Env on the surface of the retrovirus murine leukemia virus (MLV) matures by two proteolytic cleavage events mediated by cellular furin and the viral protease (1-3). Env is composed of an 80-kDa transmembrane precursor protein in the rough endoplasmic reticulum of the infected cell (4, 5). Here it trimerizes before it is routed to the cell surface for assembly with the internal Gag and GagPol precursors into virus particles in a budding process (6). The furin cleavage of Env occurs in the trans-Golgi, and it separates the surface (SU) subunit from the transmembrane (TM) (Pr15E) subunit. This cleavage also releases the viral fusion peptide at the N-terminal end of the TM. The viral protease cleavage occurs after virus assembly in the newly formed particle. It removes a 16-residuelong peptide, the R-peptide from the C terminus of the TM, forming p15E (7). Not until this second cleavage is completed is Env primed for receptor-mediated triggering into further conformations that can direct virus entry through fusion of the viral membrane with the cell membrane (8, 9). The viral protease also cleaves the Gag and GagPol precursors into their mature proteins and enzymes.The structure of the MLV Env has been studied by cryoelectron microscopy (cryo-EM) both in intact particles and as solubilized trimers (10, 11). It reveals a remarkably open structure with separated legs. The protomer of the solubilized Env is formed from three protrusions-upper, middle, and lower. Together, they encage a large central cavity, with the top pro...

“…Only low-resolution structural information is available on the unliganded HIV-1 Env trimer (20)(21)(22)(23), despite its importance as the major target for vaccine-induced neutralizing antibodies. Heavy glycosylation, unusual metastability, and structural plasticity have frustrated efforts to obtain a high-resolution structure.…”

mentioning

confidence: 99%

Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer

Mao

Wang

et al. 2013

104

120

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a "spring-loaded" conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.H uman immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus that causes AIDS (1, 2). To enter host cells, HIV-1 uses a metastable, trimeric envelope glycoprotein (Env) spike to engage cellular receptors and to fuse the viral and target cell membranes (3-5). During synthesis and folding in the endoplasmic reticulum of the virus-producing cell, the Env precursor [glycoprotein (gp)160] is heavily modified by N-linked glycosylation and assembles into trimers (6). In most HIV-1 strains, more than 27 potential N-linked glycosylation sites are present in each gp160 protomer. After further glycan processing in the Golgi apparatus, the gp160 Env trimer precursors are proteolytically cleaved and transported to the cell surface for incorporation into virions (7). Each protomer composing the trimeric Env spike thus consists of a gp120 exterior subunit and a gp41 transmembrane subunit. The sequential binding of gp120 to the target cell receptors, CD4 and chemokine receptor (either CCR5 or CXCR4), allows the metastable Env complex to transit into fusion-active conformations (3,(8)(9)(10)(11). These conformational transitions expose the hydrophobic gp41 N-terminal fusion peptide, promoting its insertion into the target cell membrane, and further permit the formation of a highly stable gp41 six-helix bundle that mediates viral-cell membrane fusion (12)(13)(14). The Env spike is the only virus-specific component potentially accessible to neutralizing antibodies and thus has evolved a protective "glycan shield" and a high degree of interstrain variability.High-resolution structures are available for monomeric HIV-1 gp120 core fragments in the CD4-boun...