The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. The envelope glycoprotein complex (Env) 4 of human immunodeficiency virus type 1 (HIV-1) comprises a trimer of receptor binding gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits on the surface of infected cells and virions. Viral entry is initiated when gp120 binds to cell-surface CD4 molecules, inducing structural changes within the gp120 core domain, and leading to the formation of the binding site for the chemokine co-receptors, CCR5 and/or CXCR4 (1-4). gp120 comprises 5 variable loops (V1-V5) that exist outside the gp120 core; V3, and to a lesser degree V1 and V2, contribute to chemokine receptor binding specificity (5-7). The sequential binding of gp120 to CD4 and chemokine receptor triggers the refolding of gp41 into a trimer of hairpins, which mediates membrane fusion (8, 9). gp41 is a class I fusion glycoprotein, being structurally homologous to the fusion glycoproteins of other retroviruses, orthomyxoviruses, paramyxoviruses, filoviruses, and coronaviruses. The gp41 ectodomain is comprised of an N-terminal fusion peptide, connected through a flexible polar segment to a coiled coil-forming amphipathic ␣-helix (N-helix), a centrally located disulfide-bonded loop, a C-terminal amphipathic ␣-helix (C-helix), and a membrane-proximal tryptophan-rich region (MPR) (10 -16). The ectodomain is anchored to the viral envelope by a C-terminally located transmembrane domain (TMD), which precedes an ϳ150-residue cytoplasmic domain.The majority of the gp41 ectodomain appears to be buried by the gp120 trimer. This model for gp41 in the context of prefusion Env is based on the findings that the epitopes of monoclonal antibodies (mAbs) encompassing the fusion peptide and polar segment (residues 521-538), disulfide bonded region (579 -613), and C-helix (644 -663), are largely occluded in pre- * This work was supported by the National Health and Medical ResearchCouncil of Australia Grants 296200 and 345413, American Foundation for AIDS Research Grant 106610-36-RGNT, and Sidaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: Env, envelope glycoprotein; HIV-1, human immunodeficiency virus type 1; N-helix, N-terminal coiled coil forming ␣-helix of gp41; C-helix, C-terminal ␣-helix of gp41; MPR, membrane proximal region; TMD, transmembrane domain; mAb, monoclonal antibody; sCD4, soluble CD4; DiO, 3,3Ј-dioctadecyloxacarbocyanine perchlorate; DiI, 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; MBP, maltose-binding protein; MALDI, matrixassisted la...
This study defines the molecular basis of the FcαRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcαRI. Biosensor analysis of mutant FcαRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcαRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcαRI was generated based on the structures of killer Ig-like receptors, which share 30–34% identity with FcαRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcαRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcαRI interaction that were observed depending on whether FcαRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.
The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2 ST gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion.
HIV-1 is spread by cell-free virions and by cell-cell viral transfer. We asked whether the structure and function of a broad neutralizing antibody (bNAb) epitope, the membrane-proximal ectodomain region (MPER) of the viral gp41 transmembrane glycoprotein, differ in cell-free and cell-cell-transmitted viruses and whether this difference could be related to Ab neutralization sensitivity. Whereas cell-free viruses bearing W666A and I675A substitutions in the MPER lacked infectivity, cell-associated mutant viruses were able to initiate robust spreading infection. Infectivity was restored to cell-free viruses by additional substitutions in the cytoplasmic tail (CT) of gp41 known to disrupt interactions with the viral matrix protein. We observed contrasting effects on cell-free virus infectivity when W666A was introduced to two transmitted/founder isolates, but both mutants could still mediate cell-cell spread. Domain swapping indicated that the disparate W666A phenotypes of the cell-free transmitted/founder viruses are controlled by sequences in variable regions 1, 2, and 4 of gp120. The sequential passaging of an MPER mutant (W672A) in peripheral blood mononuclear cells enabled selection of viral revertants with loss-of-glycan suppressor mutations in variable region 1, suggesting a functional interaction between variable region 1 and the MPER. An MPER-directed bNAb neutralized cell-free virus but not cell-cell viral spread. Our results suggest that the MPER of cell-cell-transmitted virions has a malleable structure that tolerates mutagenic disruption but is not accessible to bNAbs. In cell-free virions, interactions mediated by the CT impose an alternative MPER structure that is less tolerant of mutagenic alteration and is efficiently targeted by bNAbs.
A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384-661), that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bnAbs) in comparison to the parental form (receptor-binding domain; RBD). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S and RBD-S, and Δ123A7-S and RBDA7-S in which 7 cysteines were replaced with alanines. While all four E2-S VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells, and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bnmAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (nAbs), with three of those animals generating bnAbs against 7 genotypes. Immune serum generated by animals with nAbs mapped to major neutralization epitopes located at residues 412-420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease including cirrhosis and cancer. Broadly neutralizing antibodies that recognise the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus like particles that display epitopes recognised by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.