Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Conformational Differences between Functional Human Immunodeficiency Virus Envelope Glycoprotein Trimers and Stabilized Soluble Trimers.

Binding to the receptor CD4 triggers entry-related conformational changes in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, (gp120/gp41)3 Soluble versions of HIV-1 Env trimers (sgp140 SOSIP.664) stabilized by a gp120-gp41 disulfide bond and a change (I559P) in gp41 have been structurally characterized. Here, we use cross-linking/mass spectrometry to evaluate the conformations of functional membrane Env and sgp140 SOSIP.664. Differences were detected in the gp120 trimer association domain and C terminus and in the gp41 heptad repeat 1 (HR1) region. Whereas the membrane Env trimer exposes the gp41 HR1 coiled coil only after CD4 binding, the sgp140 SOSIP.664 HR1 coiled coil was accessible to the gp41 HR2 peptide even in the absence of CD4. Our results delineate differences in both gp120 and gp41 subunits between functional membrane Env and the sgp140 SOSIP.664 trimer and provide distance constraints that can assist validation of candidate structural models of the native HIV-1 Env trimer.IMPORTANCE HIV-1 envelope glycoprotein spikes mediate the entry of the virus into host cells and are a major target for vaccine-induced antibodies. Soluble forms of the envelope glycoproteins that are stable and easily produced have been characterized extensively and are being considered as vaccines. Here, we present evidence that these stabilized soluble envelope glycoproteins differ in multiple respects from the natural HIV-1 envelope glycoproteins. By pinpointing these differences, our results can guide the improvement of envelope glycoprotein preparations to achieve greater similarity to the viral envelope glycoprotein spike, potentially increasing their effectiveness as a vaccine.

Robustness of signal detection in cryo-electron microscopy via a bi-objective-function approach.

BACKGROUND: The detection of weak signals and selection of single particles from low-contrast micrographs of frozen hydrated biomolecules by cryo-electron microscopy (cryo-EM) represents a major practical bottleneck in cryo-EM data analysis. Template-based particle picking by an objective function using fast local correlation (FLC) allows computational extraction of a large number of candidate particles from micrographs. Another independent objective function based on maximum likelihood estimates (MLE) can be used to align the images and verify the presence of a signal in the selected particles. Despite the widespread applications of the two objective functions, an optimal combination of their utilities has not been exploited. Here we propose a bi-objective function (BOF) approach that combines both FLC and MLE and explore the potential advantages and limitations of BOF in signal detection from cryo-EM data. RESULTS: The robustness of the BOF strategy in particle selection and verification was systematically examined with both simulated and experimental cryo-EM data. We investigated how the performance of the BOF approach is quantitatively affected by the signal-to-noise ratio (SNR) of cryo-EM data and by the choice of initialization for FLC and MLE. We quantitatively pinpointed the critical SNR (~ 0.005), at which the BOF approach starts losing its ability to select and verify particles reliably. We found that the use of a Gaussian model to initialize the MLE suppresses the adverse effects of reference dependency in the FLC function used for template-matching. CONCLUSION: The BOF approach, which combines two distinct objective functions, provides a sensitive way to verify particles for downstream cryo-EM structure analysis. Importantly, reference dependency of the FLC does not necessarily transfer to the MLE, enabling the robust detection of weak signals. Our insights into the numerical behavior of the BOF approach can be used to improve automation efficiency in the cryo-EM data processing pipeline for high-resolution structural determination.

Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers.

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

SOSIP Changes Affect Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Conformation and CD4 Engagement.

The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 (sgp140) trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State 1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes with that of the well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented here suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers have utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on the conformation of Env, moving Env away from the native pretriggered Env conformation. Our studies underscore the need to acquire structural information on the pretriggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.

The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.

Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140.

UNLABELLED: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140. IMPORTANCE: A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.

Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs.

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.

Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins.

Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. IMPORTANCE: The envelope glycoproteins (Env) of HIV-1 mediate virus entry and are the sole targets of neutralizing antibodies. Understanding the way that Env promotes HIV-1 entry can expedite drug and vaccine development. By destabilizing Env, we found that it assumes an intermediate state that is functional and obligate for transitions to entry-competent conformations. Increased sampling of this state enhances the ability of HIV-1 to infect cells that express low levels of the CD4 receptor and allows the virus to evade neutralizing antibodies and small-molecule inhibitors. These findings provide new mechanistic insights into the function and inhibition of HIV-1 Env and will contribute to ongoing therapeutic and prevention efforts to combat HIV-1.