Ficolin-2 binds to HIV-1 gp120 and blocks viral infection

Ficolin-2 is a lectin complement pathway activator present in normal human plasma and usually associated with infectious diseases, but little is known about the role of ficolin-2 in human immunodeficiency virus (HIV) infection. Here, we describe our novel findings that serum ficolin-2 concentrations of 103 HIV-1 patients were much higher compared to those of 57 healthy donors. In vitro analysis showed that HIV-1 infection could enhance ficolin-2 expression. We further demonstrated that recombinant ficolin-2 protein could bind with HIV-1 envelope glycoprotein gp120, and subsequently induce complement dependent cytotoxicity. Moreover, ficolin-2 could block the entry of HIV-1 into target cells (TZM-b1 and MT-2 cells) and infection in a ficolin-2 dosedependent manner. To our knowledge, this is the first report about the protective role of ficolin-2 against HIV-1 infection and our study suggests that ficolin-2 is an important human innate immune molecule against HIV.

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Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnatural phenylalanine analogues

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the ‘Phe43 cavity’ of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.     

Peptides derived from HIV-1 gp120 co-receptor binding domain form amyloid fibrils and enhance HIV-1 infection

Amyloid fibrils play important roles in HIV-1 infection. We found peptides derived from the HIV-1 gp120 co-receptor binding region, which are defined as enhancing peptides (EPs), could form amyloid fibrils and remarkably enhance HIV-1 infection. EPs bound to the virus and promoted the interaction between HIV-1 and target cells. The antiviral efficacy of antiretroviral drugs (ARVs) was substantially impaired in the presence of EPs. Epigallocatechin gallate (EGCG) could both inhibit the formation of fibrils composed of EPs and counteract the EP-mediated enhancement of HIV-1 infection. Our findings identify viral derived amyloid fibrils that hold potential for biochemical applications.Structured summary of protein interactionsEP1 and EP1 bind by fluorescence technology (View interaction)EP2 and EP2 bind by fluorescence technology (View interaction)EP3 and EP3 bind by fluorescence technology (View interaction)SEVI and SEVI bind by fluorescence technology (View interaction)EP1 and EP1 bind by transmission electron microscopy (View interaction)EP2 and EP2 bind by transmission electron microscopy (View interaction)EP3 and EP3 bind by transmission electron microscopy (View interaction)SEVI and SEVI bind by transmission electron microscopy (View interaction)     

HIV-1 gp120对鼠海马长时程增强效应的影响

为了探讨人类免疫缺陷病毒Ⅰ型(HIV-1)的包膜糖蛋白gp120对鼠海马脑片CA1区的突触传递及可塑性的影响,应用离体脑片记录技术,记录大鼠海马CA1区的兴奋性突触后电位(excitatory postsynaptic potential,EPSP),研究了gp120对高频电刺激Schaffer侧支引起的鼠长时程增强效应(long-term potentiation,LTP)的影响.结果发现:gp120对大鼠海马CA1区LTP产生抑制作用,对其基础EPSP没有影响,而且这种抑制效应随着gp120浓度增大而增强,即具有剂量依赖性.PKA/PKC蛋白激酶抑制剂H7可以反转这种抑制效应.提示:gp120可能是通过抑制海马CA1区的LTP而参与艾滋病相关性痴呆(HIV-1 associated dementia,HAD)的形成.     

Molecular motions of human HIV-1 gp120 envelope glycoproteins

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and the coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by CD4 and antigen 17b, and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations, the details of molecular motions crucial to intervention remain elusive. We presented a comprehensive comparative analysis of dynamic behavior of gp120 in its CD4-complexed, CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops. CONCOORD computer simulation was utilized to generate ensembles of feasible protein structures, which were subsequently analyzed by essential dynamics technique to identify preferred concerted motions. The revealed collective fluctuations are dominated by complex motional modes such as rotation/twisting, flexing/closing, and shortness/elongation between or within the inner, outer, and bridging-sheet domains. An attempt has been made to relate these modes to receptor/coreceptor association and neutralization avoidance. Covariance web analysis revealed four subdomains that undergo concerted motion in gp120. The structural components in gp120 that move in concert with CD4 were also identified, which may be the suitable target for inhibitor design to interrupt CD4-gp120 interaction. The differences in B-factors between the three gp120 states revealed certain structural regions that could be related either to CD4 association or to subsequent dissociation of gp120 from gp41. These dynamics data provide new insights into the structure-function relationship of gp120 and may aid in structure-based anti-HIV vaccine design.     

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MYCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC₅₀ < 0.7 μM). MTCPP bound to the HIV-1 enveloope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to HIV-1 envelop glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anit-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.     

Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

Identification of human immunodeficiency virus type 1 (HIV-1) gp120-binding sites on scavenger receptor cysteine rich 1 (SRCR1) domain of gp340

Background

gp340, a member of scavenger receptor cysteine rich family encoded by Deleted in Malignant Brain Tumors 1 (DMBT1), is an important component in innate immune defense. The first scavenger receptor cysteine rich domain (SRCR1) of gp340 has been shown to inhibit HIV-1 infection through binding to the N-terminal flank of the V3 loop of HIV-1 gp120.

Results

Through homology modeling and docking analysis of SRCR1 to a gp120-CD4-X5 antibody complex, we identified three loop regions containing polar or acidic residues that directly interacted with gp120. To confirm the docking prediction, a series of over-lapping peptides covering the SRCR1 sequence were synthesized and analyzed by gp120-peptide binding assay. Five peptides coincide with three loop regions showed the relative high binding index. An alanine substitution scan revealed that Asp34, Asp35, Asn96 and Glu101 in two peptides with the highest binding index are the critical residues in SRCR1 interaction with gp120.

Conclusion

We pinpointed the vital gp120-binding regions in SRCR1 and narrowed down the amino acids which play critical roles in contacting with gp120.     

Role of Curdlan Sulfate in the Binding of HIV-1 gp120 to CD4 Molecules and the Production of gp120-Mediated TNF-α

To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)-1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF-α by gp120-stimulated macrophages (which promotes HIV-1 replication). CRDS treatment of cells not only inhibited the binding of HIV-1 gp120 to CD4⁺ cells, but also inhibited TNF-α production induced by gp120. Inhibition of HIV-1 infection by CRDS may be related to these two actions.     

The interaction of a glycosaminoglycan heparin,with HIV-1 major envelope glycoprotein

We demonstrate in vitro the occurence of a specific but low-affinity interaction between soluble tetrameric rgp160 or soluble monomeric or tetrameric rgp120 and heparin-agarose (HA). This interaction is saturable, pH and temperature-dependent, and can be inhibited by soluble heparin, but not by soluble dextran. In buffer supplemented with 10 mM CaCl₂, the C₅₀ of soluble heparin, i.e., the concentration of soluble heparin which leads to 50% inhibition of the binding of [¹²⁵I]rgp160 or [¹²⁵I]rgp120 to HA, is 1.1. · 10^?4 disaccharidic molar concentration for rgp160 and 3.2 · 10^?4 disaccharidic molar concentration for rgp120, which indicates low-affinity interactions. Upon chromatography on HA, [¹²⁵I]rgp160 is repeatedly eluted as a retarded fraction when compared to the elutions volume of [¹²⁵I]rgp160-soluble heparin complex. Under the same experimental conditions, [¹²⁵I]rgp120 is also eluted, but as a less retarded fraction than [¹²⁵I]rgp160. Taken together, these results suggest that, at least part of the described anti HIV-1 activity of heparin might be mediated by interaction with HIV-1 major envelope glycoprotein.     

Prediction of the secondary structure of HIV-1 gp120

The secondary structure of HIV-1 gp120 was predicted using multiple alignment and a combination of two independent methods based on neural network and nearest-neighbor algorithms. The methods agreed on the secondary structure for 80% of the residues in BH10 gp120. Six helices were predicted in HIV strain BH10 gp120, as well as in 27 other HIV-1 strains examined. Two helical segments were predicted in regions displaying profound sequence variation, one in a region suggested to be critical for CD4 binding. The predicted content of helix, β-strand, and coil was consistent with estimates from Fourier transform infrared spectroscopy. The predicted secondary structure of gp120 compared well with data from NMR analysis of synthetic peptides from the V3 loop and the C4 region. As a first step towards modeling the tertiary structure of gp120, the predicted secondary structure may guide the design of future HIV sub-unit vaccine candidates. © 1996 Wiley-Liss, Inc.     

Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity

The interaction of the HIV-1 fusion protein gp120 with its cellular receptor CD4 represents a crucial step of the viral infection process, thus rendering gp120 a promising target for the intervention with anti-HIV drugs. Naturally occurring mutations of gp120, however, can decrease its affinity for anti-infective ligands like therapeutic antibodies or soluble CD4. To understand this phenomenon on a structural level, we performed molecular dynamics simulations of two gp120 variants (termed gp120_3-2 and gp120_2-1), which exhibit a significantly decreased binding of soluble CD4. In both variants, the exchange of a nonpolar residue byglutamate was identified as an important determinant for reduced binding. However, those glutamates are located at different sequence positions and affect different steps of the recognition process: E471 in gp120_3-2 predominantly affects the CD4-bound conformation, whereas E372 in gp120_2-1 mainly modulates the conformational sampling of free gp120. Despite these differences, there exists an interesting similarity between the two variants: both glutamates exert their function by modulating the conformation and interactions of glycine-rich motifs (G366–G367, G471–G473) resulting in an accumulation of binding incompetent gp120 conformations or a loss of intermolecular gp120–CD4 hydrogen bonds. Thus, the present data suggests that interference with the structure and dynamics of glycine-rich stretches might represent a more widespread mechanism, by which gp120 mutations reduce binding affinity. This knowledge should be helpful to predict the resistance of novel gp120 mutations or to design gp120–ligands with improved binding properties.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:41     

Cholesterol-dependent modulation of the toxicity of HIV-1 coat protein gp120 in human neuroblastoma cells

The human immunodeficiency virus type 1 (HIV-1) coat glycoprotein gp120 binds to its (co)receptors and orchestrates cell entry by the direct fusion of viral and target cell membranes. Here, we modulated membrane fluidity of human neuroblastoma CHP100 cells by modulating their cholesterol content, and investigated the ability of gp120 to induce cell death in comparison with the untreated cells. We show that in normal CHP100 cells gp120 induces necrosis by: (i) increased cyclooxygenase and 5-lipoxygenase activity, and metabolites generated thereof (prostaglandin E2 and leukotriene B4, respectively); (ii) increased membrane lipoperoxidation; and (iii) increased mitochondrial uncoupling. These events were triggered by a rapid increase in intracellular calcium, and in cholesterol-depleted cells engaged CXCR4 chemokine receptors. The intracellular calcium chelator EGTA-AM protected CHP100 cells almost completely against the toxic effects of gp120. However, gp120-induced necrosis and related biochemical changes were negligible in cholesterol-enriched, and significantly enhanced in cholesterol-depleted, CHP100 cells exposed to the viral glycoprotein under the same experimental conditions. Taken together, these results suggest that membrane fluidity may control the neurotoxic effects of HIV-1 glycoprotein gp120.     

Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.     

The V1/V2 loop of HIV-1 gp120 is necessary for Tat binding and consequent modulation of virus entry

Preventing cell entry of human immunodeficiency virus 1 (HIV-1) is of interest for the development of innovative therapies. We previously reported a specific interaction between HIV-1 envelope glycoprotein 120 (gp120) and Tat at the cell surface, which enhances virus attachment and entry. We also identified a gp120-mimicking peptide, CT319, that competes with gp120 for Tat binding, thus inhibiting HIV-1 infection. Here we report a molecular dissection of gp120 regions involved in this mechanism. Our findings identify the V1/V2 loop of gp120 as involved in Tat binding, and define this interaction as functionally relevant for HIV-1 entry into host cells.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

Pharmacokinetics of an HIV-1 gp120-specific chimeric antibody in patients with HIV-1 disease

The pharmacokinetics of mouse V/human C (1,) chimeric monoclonal antibody CGP47 439 specific for the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) was studied in patients with stage IV HIV-1 disease in an open-labeled phase I/IIA trial. Twelve male patients were enrolled and nine completed the study. Patients were divided into three groups according to the extent of CGP 47 439 to bind to gp120 from their viral isolates: undetectable for group 1, modestly reactive for group 2, and strongly reactive for group 3. A first dose of 1, 10, or 25 mg was administered by intravenous infusion to group 1, group 2 and group 3 patients, respectively. The patients then received seven doses of 50, 100, or 200 mg, respectively, every three weeks. CGP 47 439 serum concentrations were determined by an ELISA using monoclonal antibody AB19-4 specific for the idiotope of CGP 47 439. Half an hour after infusion only 25.5–36.1% of the administered antibody was found in the serum, reflecting its rapid distribution in the extravascular space and possibly binding to gp120 antigen in some of the patients. The terminal elimination half-life (T_1/2) was 16.2 days in group 1 patients, 9.7 days in group 2 and in group 3 patients 7.5 days and 9.1 days. An antibody response to CGP 47 439 was not a factor in determining elimination rates, because only very low and transient responses were found in three patients. These results suggest that the reactivity of CGP 47 439 with HIV-1 gp120 contributed to its elimination in HIV-1 infected patients.Abbreviations AIDS aquired immune deficiency syndrome - ARC AIDS-related complex - HIV-1 human immune deficiency virus type 1 - gp120 envelope glycoprotein with 120 KD molecular weight - V3 variable domain of gp120 - PND principle neutralizing determinant of gp120 - IgG immunoglobulin G - CD4⁺ lymphocytes: lymphocytes expressing the CD4 marker V_H and V_L variable heavy and variable light chain region of an antibody - C₁ and C_K constant heavy chain region of gamma l and constant K light chain region of an antibody - anti-id anti-idiotypic - AUC area under curve - T_1/2 terminal elimination half-life - ELISA enzyme-linked imuno sorbent assay - PBS phosphate buffered saline - NP-40 detergent - CDC center of disease control - GMP good manufacturing practice     

Construction of Peptides Mimicking a HIV-1 gp41 Epitope Recognized by Virus-Neutralizing Antibodies 2F5

A phage peptide library was screened with virus-neutralizing monoclonal antibodies (MAb) 2F5 which recognize a conserved epitope of HIV-1 gp41. Phages that expose peptides specifically binding with MAb 2F5 were selected by ELISA. Amino acid sequence analysis revealed homology to region 662–671 of HIV-1 HB10 gp160 for most peptides. The major role in recognition was ascribed to Asp-664, Lys-665, and Trp-666. The epitope-mimicking peptides were tested for immunogenicity. Antibodies to gp41 were detected in the serum of immunized rabbits.