HIV-1 envelope glycoprotein-mediated fusion and pathogenesis: Implications for therapy and vaccine development

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N-and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp 632 located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys 574 in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys 574 plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp 632 has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp 632 substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys 574 mutations, nonconserved substitutions of Asp 632 also severely impaired the ␣-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp 632 significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N-and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.Infection with human immunodeficiency virus type 1 (HIV-1) is mediated by its envelope glycoprotein (Env), a type I transmembrane protein which is originally synthesized as the single, glycosylated, polyprotein precursor gp160 and subsequently cleaved by a cellular protease to yield gp120 and gp41 subunits (13,14,20,46,48). Upon binding of the HIV-1 Env surface subunit gp120 to the cell receptor CD4 and subsequently to a coreceptor (CCR5 or CXCR4), its transmembrane subunit gp41 is released to mediate fusion of viral and cellular membranes (20,25,54). Structurally, HIV-1 gp41 consists of extracellular, transmembrane, and cytoplasmic domains (Fig. 1A). Its extracellular domain (ectodomain) contains four major functional regions: a hydrophobic, glycine-rich fusion peptide; an N-terminal heptad repeat (NHR) (also called HR1), a C-terminal heptad repeat (CHR) (also called HR2), and a tryptophan-rich region. In the early 1990s, several peptides derived from the NHR (N peptides) and CHR (C peptides) were found to have potent anti-HIV activity (30,43,68,69). Although their mechanism of action was not known at that time, the unprecedented anti-HIV activity of these peptides opened a new avenue for developing antiviral drugs. A C peptide known as T20 (brand name, Fuzeon) has been successfully developed as a novel class of anti-HIV drugs for clinical use (36,37,50).The findin...

Section: Discussionmentioning

confidence: 99%

“…Upon binding of the HIV-1 Env surface subunit gp120 to the cell receptor CD4 and subsequently to a coreceptor (CCR5 or CXCR4), its transmembrane subunit gp41 is released to mediate fusion of viral and cellular membranes (20,25,54). Structurally, HIV-1 gp41 consists of extracellular, transmembrane, and cytoplasmic domains (Fig.…”

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confidence: 99%

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

Liu

et al. 2008

“…To validate the antibody-virus washout assay, we first incubated the known neutralizing peptide ligand, T-20, with both lab-adapted virus, HXBc2, and the primary isolate, JR-FL (71). Because this peptide potently inhibits virus entry by binding to the receptor activated transitional intermediate that exposes the HR1 coiled-coil region of gp41, but only after receptor engagement (12,25,30,31,64), we used this compound to confirm complete washout from the system of a neutralizing ligand that cannot access its determinant in the absence of receptor-induced activation. Accordingly, the virus was incubated either with buffer alone or with the T-20 peptide at a concentration 200-fold in excess of its 50% inhibitory concentration (IC 50 ).…”

Section: F5 and 4e10 Bind Inefficiently To Cleaved Full-length Jr-fmentioning

confidence: 99%

Direct Antibody Access to the HIV-1 Membrane-Proximal External Region Positively Correlates with Neutralization Sensitivity

Chakrabarti

Walker

Guenaga

et al. 2011

On the prereceptor-engaged HIV-1 envelope glycoprotein (Env) spike, epitope access by the membraneproximal external region (MPER)-directed broadly neutralizing antibodies 2F5 and 4E10 remains unresolved. Data on binding to cell surface Env and entry data using primary isolates suggest inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and slow kinetics of shedding induced by 2F5 and 4E10 indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed in their antibody-bound state (or at least sampled) prior to receptor/coreceptor engagement or if receptor interactions both expose and form the MPER epitopes, presumably in the putative prefusion transitional intermediate. Here, we performed antibody-virus "washout experiments" using both lab-adapted and a panel of clade B primary isolates to analyze MPER accessibility. The neutralization activity of 2F5 and 4E10 against lab-adapted viruses and sensitive and moderately resistant viruses was largely unaffected by relatively rapid antibody-virus washing, suggesting direct interaction with the "static" spike. However, for more neutralization-resistant viruses, the 2F5 and 4E10 antibodies could neutralize only under the "no antibody-virus wash" conditions, implying that the MPER epitopes were not accessible prior to receptor engagement. Accessibility in the washout conditions could be precisely predicted by the relative resistance to neutralization in a standard neutralization format. These data are consistent with a model in which the local MPER antibody epitope conformations may be sampled on the native spike but are occluded to antibody by local steric or distal quaternary constraints adopted by highly resistant HIV-1 isolates.The HIV-1 gp160 envelope glycoprotein (Env) precursor is cleaved by cellular furins to generate the noncovalently associated gp120 and gp41 trimeric Env complex. The gp120 subunit binds to the primary receptor, CD4, and, following conformational changes, also interacts with the CCR5 coreceptor. The gp41 trans-membrane Env then mediates virus-to-cell membrane fusion, resulting in the entry of viral genomic information into the target cell (8,11,14,24,(64)(65)(66). The genetic variability of Env, coupled with the inaccessibility of conserved epitopes, makes the generation of antibodies capable of neutralizing a broad array of primary isolates (i.e., broadly neutralizing) a substantial challenge. Viral entry into cells can be blocked in vitro by relatively rare broadly neutralizing antibodies that are elicited during the course of natural infection. Since viral half-life in vivo is relatively brief (t 1/2 , 30 to 60 min) (40, 57), HIV-1 neutralizing antibodies need to act rapidly to inactivate infectious virus. Hence, most in vitro neutralization assays, which assess the ability of antibody to interfere with HIV entry, are traditionally performed with approximately 1-h incubation times of antibody-virus (41,42,70).Neutralizing antibodies can either ef...

“…The conformational changes in gp120 involve the movement of the first, second, and third variable loops (V1, V2, and V3 loops) that normally shield the coreceptor binding site (68,69,78). Additional conformational changes in the trimeric complex lead to the exposure of hydrophobic fusion peptides at the N termini of the gp41 subunits, culminating in the fusion of the viral and cellular membranes (13,16,31,36,75).The individual subunits have proven unsuccessful as vaccines, presumably because they do not resemble the functional Env complex present on infectious virus particles. We and others have generated stabilized trimeric Env, yielding incremental to modest improvements of Env immunogenicity (4, 4-6, 21, 23, 62, 65, 80, 82).…”

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confidence: 99%

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confidence: 99%

Optimization of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins with V1/V2 Deleted, Using Virus Evolution

Bontjer

Land

Eggink

et al. 2009

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (Env) is the principal target of vaccine research aimed at raising an antiviral humoral immune response. The isolation of a small number of broadly active neutralizing antibodies from HIV-infected individuals serves as a rationale for the search for vaccines that elicit such antibodies. Although many Env-based HIV-1 vaccines have been tested in preclinical and clinical studies, so far none has raised broadly reactive antibodies that could neutralize diverse primary virus isolates. Thus, relatively straightforward vaccine strategies that worked for other pathogens-for example, the use of unmodified surface antigens-have been explored without satisfactory results for HIV-1, emphasizing the necessity for more-sophisticated vaccine design.The functional HIV-1 Env complex, which mediates viral entry into CD4 ϩ host cells, is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The attachment of HIV-1 to a target cell is followed by the fusion of the viral and cellular membranes. First, gp120 binds to the CD4 receptor, a process that induces conformational changes to create and expose the coreceptor binding site (71, 76). The...