High Mannose-binding Lectin with Preference for the Cluster of α1–2-Mannose from the Green Alga Boodlea coacta Is a Potent Entry Inhibitor of HIV-1 and Influenza Viruses

The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1–2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1–2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1–2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 108m−1) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent.

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Man-Specific, GalNAc/T/Tn-Specific and Neu5Ac-Specific Seaweed Lectins as Glycan Probes for the SARS-CoV-2 (COVID-19) Coronavirus

Marine Drugs ◽

10.3390/md18110543 ◽

2020 ◽

Vol 18 (11) ◽

pp. 543

Author(s):

Annick Barre ◽

Els J.M. Van Damme ◽

Mathias Simplicien ◽

Hervé Benoist ◽

Pierre Rougé

Keyword(s):

Influenza Virus ◽

Antiviral Agents ◽

Host Cells ◽

Carbohydrate Binding ◽

Complex Type ◽

Enveloped Viruses ◽

High Mannose ◽

Hiv 1 ◽

Biomedical Interest

Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells—in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.

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Monitoring Early Fusion Dynamics of Human Immunodeficiency Virus Type 1 at Single-Molecule Resolution

Journal of Virology ◽

10.1128/jvi.00053-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 7022-7033 ◽

Cited By ~ 38

Author(s):

Terrence M. Dobrowsky ◽

Yan Zhou ◽

Sean X. Sun ◽

Robert F. Siliciano ◽

Denis Wirtz

Keyword(s):

Human Immunodeficiency Virus ◽

Tensile Strength ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Single Molecule ◽

Viral Envelope ◽

Host Cells ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The fusion of human immunodeficiency virus type 1 (HIV-1) to host cells is a dynamic process governed by the interaction between glycoproteins on the viral envelope and the major receptor, CD4, and coreceptor on the surface of the cell. How these receptors organize at the virion-cell interface to promote a fusion-competent site is not well understood. Using single-molecule force spectroscopy, we map the tensile strengths, lifetimes, and energy barriers of individual intermolecular bonds between CCR5-tropic HIV-1 gp120 and its receptors CD4 and CCR5 or CXCR4 as a function of the interaction time with the cell. According to the Bell model, at short times of contact between cell and virion, the gp120-CD4 bond is able to withstand forces up to 35 pN and has an initial lifetime of 0.27 s and an intermolecular length of interaction of 0.34 nm. The initial bond also has an energy barrier of 6.7 kB T (where kB is Boltzmann's constant and T is absolute temperature). However, within 0.3 s, individual gp120-CD4 bonds undergo rapid destabilization accompanied by a shortened lifetime and a lowered tensile strength. This destabilization is significantly enhanced by the coreceptor CCR5, not by CXCR4 or fusion inhibitors, which suggests that it is directly related to a conformational change in the gp120-CD4 bond. These measurements highlight the instability and low tensile strength of gp120-receptor bonds, uncover a synergistic role for CCR5 in the progression of the gp120-CD4 bond, and suggest that the cell-virus adhesion complex is functionally arranged about a long-lived gp120-coreceptor bond.

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Targeting HIV-1 Envelope Proteins Using a Fragment Discovery All-Atom Computational Algorithm

Current Enzyme Inhibition ◽

10.2174/1573408012666160725095854 ◽

2017 ◽

Vol 13 (1) ◽

pp. 20-26

Author(s):

Michael H. Peters

Keyword(s):

Peptide Bond ◽

Envelope Proteins ◽

Peptide Inhibitors ◽

Viral Envelope ◽

Host Cells ◽

Heptad Repeat ◽

Computational Time ◽

Viral Inhibition ◽

Initial Testing ◽

Hiv 1

Introduction: HIV viral envelope proteins are targets for small inhibitor molecules aimed at disrupting the cellular entry process. Potential peptide-class inhibitor molecules (rDNA drugs) have been previously identified, with mixed results, through biomimicry and phage display experimental methods. Here we describe a new approach based on computational fragment discovery. The method has the potential to not only optimize peptide binding affinity but also to rapidly produce alternative inhibitors against mutated strains. Methods: A comprehensive, all-atom implicit solvent method is used to bombard the C-heptad repeat unit of HIV-1 target envelope protein GP41 with single Damino acid residues as they exist in their native state. A nascent peptide computational search process then identifies potential favorable sequences of attached ligands based on four peptide bond criteria. Finally, dynamic simulations of nascent peptides attached to host targets help refine potential peptide inhibitors for experimental HIV-1 challenge assays and testing. Results and Discussion: Initial testing of the method was done using 64,000 total ligands of D-amino acid residues at a total computational time of 0.05 microseconds per ligand, which resulted in several thousand attached ligands. Peptide bond criteria search employing three of the four bond constraints with a tolerance of 20 percent, resulted in four potential peptide inhibitors of 5 to 6 residues in length. Only one of the four peptides demonstrated IC50 values and partial viral inhibition based on cell challenge assays using CEM-SS host cells. That peptide inhibitor also computationally demonstrated longtime attachment and stability to a helical groove in its C-heptad target. This initial testing of peptide fragment discovery against HIV-1 has helped us refine the protocols and identify key areas of improvement. Conclusion: Our methods demonstrate the potential to design efficient peptide inhibitors to viral target proteins based on an all-atom dynamic simulation and using a ligand library as fragments of potential nascent peptides. Our methods can be greatly improved through the use of higher numbers of ligands, increased time of bombardment, and tighter constraints on the peptide bond search step. Our method may be important in the need to rapidly respond to target mutations and to advance multiple targeting methods based multiple peptide inhibitors.

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Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate

10.1101/2020.04.29.068775 ◽

2020 ◽

Author(s):

Mark S. Ladinsky ◽

Priyanthi N.P. Gnanapragasam ◽

Zhi Yang ◽

Anthony P. West ◽

Michael S Kay ◽

...

Keyword(s):

Electron Tomography ◽

Intermediate Stage ◽

Viral Envelope ◽

Host Cells ◽

Target Cells ◽

Virus Infectivity ◽

Viral Fusion ◽

Fusion Inhibitors ◽

Viral Egress ◽

Hiv 1

AbstractFusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by electron microscopy (EM). To capture fusion events for EM, the process must be slowed or stopped by trapping Env-receptor binding at an intermediate stage. Here we describe using fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.

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Staphylococcus aureus Leukocidin LukED and HIV-1 gp120 Target Different Sequence Determinants on CCR5

mBio ◽

10.1128/mbio.02024-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 11

Author(s):

Kayan Tam ◽

Megan Schultz ◽

Tamara Reyes-Robles ◽

Bénédicte Vanwalscappel ◽

Joshua Horton ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Chemokine Receptors ◽

Point Mutations ◽

Cell Lysis ◽

Vital Role ◽

Viral Envelope ◽

Host Cells ◽

Mediate Cytotoxicity ◽

Necessary And Sufficient ◽

Hiv 1

ABSTRACT Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5. IMPORTANCE The bicomponent pore-forming toxins are thought to play a vital role in the success of Staphylococcus aureus as a mammalian pathogen. One of the leukocidins, LukED, is necessary and sufficient for lethality in mice. At the molecular level, LukED causes cell lysis through binding to specific cellular receptors. CCR5 is one of the receptors targeted by LukED and is the major coreceptor for CCR5-tropic HIV-1. While the molecular interaction of CCR5 and HIV-1 is well characterized, the means by which LukED interacts with CCR5 is less clear. In this study, we demonstrated that receptor specificity is conferred through unique interactions between key domains on CCR5 and LukE. Although HIV-1 and LukED target the same receptor, our data demonstrated that they interact with CCR5 differently, highlighting the molecular complexity of host-pathogen interactions.

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Discoveries and developments of CXCR4-targeted HIV-1 entry inhibitors

Experimental Biology and Medicine ◽

10.1177/1535370220901498 ◽

2020 ◽

Vol 245 (5) ◽

pp. 477-485

Author(s):

Chaozai Zhang ◽

Ruohan Zhu ◽

Qizhi Cao ◽

Xiaohong Yang ◽

Ziwei Huang ◽

...

Keyword(s):

Viral Replication ◽

Structural Features ◽

Viral Envelope ◽

Host Cells ◽

Target Cells ◽

Rapid Progression ◽

Genomic Integration ◽

Entry Inhibitors ◽

Anti Hiv ◽

Hiv 1

The chemokine receptor CXCR4 is required for the entry of human immunodeficiency virus type 1 (HIV-1) into target cells and its expression correlates with more profound pathogenicity, rapid progression to acquired immunodeficiency syndrome (AIDS), and greater AIDS-related mortality. There is still no cure for AIDS and no method for preventing or eradicating HIV-1 infection. HIV-1 entry begins with the interaction of the viral envelope glycoprotein gp120 and the primary receptor CD4, and subsequently with the coreceptors, CCR5 or CXCR4, on the host cells. Blocking the interaction of HIV-1 and its coreceptors is therefore a promising strategy for developing new HIV-1 entry inhibitors. This approach has a dual benefit, as it prevents HIV-1 infection and progression while also targeting the reservoirs of HIV-1 infected, coreceptor positive macrophages and memory T cells. To date, multiple classes of CXCR4-targeted anti-HIV-1 inhibitors have been discovered and are now at different preclinical and clinical stages. In this review, we highlight the studies of CXCR4-targeted small-molecule and peptide HIV-1 entry inhibitors discovered during the last two decades and provide a reference for further potential HIV-1 exploration in the future. Impact statement This minireview summarized the current progress in the identification of CXCR4-targeted HIV-1-entry inhibitors based on discovery/developmental approaches. It also provided a discussion of the inhibitor structural features, antiviral activities, and pharmacological properties. Unlike other reviews on anti-HIV-1 drug development, which have generally emphasized inhibitors that target intracellular viral replication and host genomic integration, this review focused on the drug discovery approaches taken to develop viral-entry inhibitors aimed at disturbing the initial step of viral interaction with uninfected host cells and preventing the subsequent viral replication/genomic integration. This review amalgamated recently published and important work on bivalent CXCR4-targeted anti-HIV-1-entry candidates/conjugates, discussed the research challenges faced in developing drugs to prevent and eradicate HIV-1 infection, and provided a perspective on strategies that can lead to future drug discoveries. The findings and strategies summarized in this review will be of interest to investigators throughout the microbiological, pharmaceutical, and translational research communities.

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

Journal of Virology ◽

10.1128/jvi.01709-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 6

Author(s):

Luis R. Castillo-Menendez ◽

Hanh T. Nguyen ◽

Joseph Sodroski

Keyword(s):

Human Immunodeficiency Virus ◽

Envelope Glycoprotein ◽

Coiled Coil ◽

Viral Envelope ◽

Host Cells ◽

Heptad Repeat ◽

Envelope Glycoproteins ◽

Immunodeficiency Virus ◽

Functional Membrane ◽

Hiv 1

ABSTRACTBinding 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.IMPORTANCEHIV-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.

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Removal of two high-mannose N-linked glycans on gp120 renders human immunodeficiency virus 1 largely resistant to the carbohydrate-binding agent griffithsin

Journal of General Virology ◽

10.1099/vir.0.033092-0 ◽

2011 ◽

Vol 92 (10) ◽

pp. 2367-2373 ◽

Cited By ~ 28

Author(s):

Xin Huang ◽

Wei Jin ◽

George E. Griffin ◽

Robin J. Shattock ◽

Qinxue Hu

Keyword(s):

Human Immunodeficiency Virus ◽

Neutralizing Antibodies ◽

Site Directed Mutagenesis ◽

Carbohydrate Binding ◽

Immunodeficiency Virus ◽

Galanthus Nivalis ◽

High Mannose ◽

Topical Microbicides ◽

Marked Resistance ◽

Hiv 1

High-mannose N-linked glycans recognized by carbohydrate-binding agents (CBAs) are potential targets for topical microbicides. To better understand the mechanisms by which CBAs inhibit human immunodeficiency virus (HIV)-1 infection at the molecular level, we systematically analysed the contribution of site-specific glycans to the anti-HIV activity of CBAs by site-directed mutagenesis. Our results demonstrate that a single deglycosylation at N295 or N448 in a range of primary and T-cell-line-adapted HIV-1 isolates resulted in marked resistance to griffithsin (GRFT) but maintained the sensitivity to cyanovirin (CV-N), Galanthus nivalis agglutinin (GNA) and a range of neutralizing antibodies. Unlike CV-N and GNA, the interaction between GRFT and gp120 appeared to be dependent on the specific trimeric ‘sugar tower’ including N295 and N448. This was further strengthened by the results of GRFT–Env binding experiments. Our study identifies GRFT-specific gp120 glycans and may provide information for the design of novel CBA antiviral strategies.

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Quaternary Interaction of the HIV-1 Envelope Trimer with CD4 and Neutralizing Antibodies

Viruses ◽

10.3390/v13071405 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1405

Author(s):

Qingbo Liu ◽

Peng Zhang ◽

Paolo Lusso

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Neutralizing Antibodies ◽

Current Knowledge ◽

Viral Envelope ◽

Host Cells ◽

Initial Contact ◽

Cd4 Receptor ◽

Cd4 Binding Site ◽

Hiv 1

The entry of HIV-1 into host cells is initiated by the interaction of the viral envelope (Env) spike with the CD4 receptor. During this process, the spike undergoes a series of conformational changes that eventually lead to the exposure of the fusion peptide located at the N-terminus of the transmembrane glycoprotein, gp41. Recent structural and functional studies have provided important insights into the interaction of Env with CD4 at various stages. However, a fine elucidation of the earliest events of CD4 contact and its immediate effect on the Env conformation remains a challenge for investigation. Here, we summarize the discovery of the quaternary nature of the CD4-binding site in the HIV-1 Env and the role of quaternary contact in the functional interaction with the CD4 receptor. We propose two models for this initial contact based on the current knowledge and discuss how a better understanding of the quaternary interaction may lead to improved immunogens and antibodies targeting the CD4-binding site.

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Man-Specific Lectins from Plants, Fungi, Algae and Cyanobacteria, as Potential Blockers for SARS-CoV, MERS-CoV and SARS-CoV-2 (COVID-19) Coronaviruses: Biomedical Perspectives

Cells ◽

10.3390/cells10071619 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1619

Author(s):

Annick Barre ◽

Els J. M. Van Van Damme ◽

Mathias Simplicien ◽

Sophie Le Le Poder ◽

Bernard Klonjkowski ◽

...

Keyword(s):

Host Cells ◽

Carbohydrate Binding ◽

Binding Domains ◽

Mannose Binding ◽

Virion Envelope ◽

Coronavirus Infection ◽

High Mannose ◽

And Control ◽

Legume Lectins ◽

S Proteins

Betacoronaviruses, responsible for the “Severe Acute Respiratory Syndrome” (SARS) and the “Middle East Respiratory Syndrome” (MERS), use the spikes protruding from the virion envelope to attach and subsequently infect the host cells. The coronavirus spike (S) proteins contain receptor binding domains (RBD), allowing the specific recognition of either the dipeptidyl peptidase CD23 (MERS-CoV) or the angiotensin-converting enzyme ACE2 (SARS-Cov, SARS-CoV-2) host cell receptors. The heavily glycosylated S protein includes both complex and high-mannose type N-glycans that are well exposed at the surface of the spikes. A detailed analysis of the carbohydrate-binding specificity of mannose-binding lectins from plants, algae, fungi, and bacteria, revealed that, depending on their origin, they preferentially recognize either complex type N-glycans, or high-mannose type N-glycans. Since both complex and high-mannose glycans substantially decorate the S proteins, mannose-specific lectins are potentially useful glycan probes for targeting the SARS-CoV, MERS-CoV, and SARS-CoV-2 virions. Mannose-binding legume lectins, like pea lectin, and monocot mannose-binding lectins, like snowdrop lectin or the algal lectin griffithsin, which specifically recognize complex N-glycans and high-mannose glycans, respectively, are particularly adapted for targeting coronaviruses. The biomedical prospects of targeting coronaviruses with mannose-specific lectins are wide-ranging including detection, immobilization, prevention, and control of coronavirus infection.

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