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
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| S-EPMC136400 | biostudies-literature
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.
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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
Project description:We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases.
| S-EPMC1217081 | biostudies-other