Proteolytic processing of the human immunodeficiency virus envelope glycoprotein precursor decreases conformational flexibility.
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ABSTRACT:
SUBMITTER: Haim H
PROVIDER: S-EPMC3554131 | biostudies-literature | 2013 Feb
REPOSITORIES: biostudies-literature
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Proteolytic processing of the human immunodeficiency virus envelope glycoprotein precursor decreases conformational flexibility.
Haim Hillel H Salas Ignacio I Sodroski Joseph J
Journal of virology 20121121 3
The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentially a ...[more]
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Project description: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.
| S-EPMC6340038 | biostudies-literature
Project description: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.
| S-EPMC5289478 | biostudies-literature
Project description:The design of an efficient human immunodeficiency virus (HIV) immunogen able to generate broad neutralizing antibodies (NAbs) remains an elusive goal. As more data emerge, it is becoming apparent that one important aspect of such an immunogen will be the proper representation of the envelope protein (Env) as it exists on native virions. Important questions that are yet to be fully addressed include what factors dictate Env processing, how different Env forms are represented on the virion, and ultimately how these issues influence the development and efficacy of NAbs. Recent data have begun to illuminate the extent to which changes in gp41 can impact the overall structure and neutralizing sensitivity of Env. Here, we present evidence to suggest that minor mutations in gp120 can significantly impact Env processing. We analyzed the gp120 sequences of 20 env variants that evolved in multiple macaques over 8 months of infection with simian/human immunodeficiency virus 89.6P. Variant gp120 sequences were subcloned into gp160 expression plasmids with identical cleavage motifs and gp41 sequences. Cells cotransfected with these plasmids and delta env genomes were able to produce competent virus. The resulting pseudoviruses incorporated high levels of Env onto virions that exhibited a range of degrees of virion-associated Env cleavage (15 to 40%). Higher levels of cleavage correlated with increased infectivity and increased resistance to macaque plasma, HIV immunoglobulin, soluble CD4, and human monoclonal antibodies 4E10, 2F5, and b12. Based on these data, we discuss a model whereby changes in gp120 of 89.6P impact Env processing and thereby mediate escape from a range of neutralizing agents.
| S-EPMC2169095 | biostudies-literature
Project description:The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.
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Project description:The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.
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Project description:Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.
| S-EPMC7492737 | biostudies-literature
Project description: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.
| S-EPMC5974486 | biostudies-literature
Project description:Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.
| S-EPMC2791628 | biostudies-literature
Project description:In this study, we have investigated the effect of specific mutations in human immunodeficiency virus type 1 (HIV-1) envelope (Env) on antibody production in an effort to improve humoral immune responses to this glycoprotein by DNA vaccination. Mice were injected with plasmid expression vectors encoding HIV Env with modifications in regions that might affect this response. Elimination of conserved glycosylation sites did not substantially enhance humoral or cytotoxic-T-lymphocyte (CTL) immunity. In contrast, a modified gp140 with different COOH-terminal mutations intended to mimic a fusion intermediate and stabilize trimer formation enhanced humoral immunity without reducing the efficacy of the CTL response. This mutant, with deletions in the cleavage site, fusogenic domain, and spacing of heptad repeats 1 and 2, retained native antigenic conformational determinants as defined by binding to known monoclonal antibodies or CD4, oligomer formation, and virus neutralization in vitro. Importantly, this modified Env, gp140 Delta CFI, stimulated the antibody response to native gp160 while it retained its ability to induce a CTL response, a desirable feature for an AIDS vaccine.
| S-EPMC137015 | biostudies-literature