HIV gp120 induced gene expression signatures in vaginal epithelial cells

Gp120 is the envelope protein of HIV which binds to CD4 independent proteins on vaginal epithelial cells. HIV-gp120 has been reported to modulate gene expression in several cell types. How this interaction may alter the physiologic vaginal milieu during the earliest stages of vaginal transmission of HIV, is currently unknown. Vaginal epithelial cells were treated with HIV-gp120, and a global snapshot of changes in gene expression profiles, were unraveled by microarray analysis. The differentially expressed genes were involved in diverse cellular functions. Genes of immunomodulatory processes and induction of proteases were highly enriched. We propose that the induction of inflammation and proteases may act in concert to weaken the vaginal epithelium, making it more permeable to viral entry. Identification of the gene signatures involved in vaginal-HIV dialogue would aid in understanding the environ induced by HIV itself, as the virus invades and gains entry into its host.     

Correlated mutations at gp120 positions 322 and 440: implications for gp120 structure

Analysis of V3 and C4 sequences of HIV-1 reveals correlated mutations at gp120 positions 322 and 440, and a very strong preference for a positively charged residue at position 440 when position 322 is negatively charged. This observation suggests that these two residues are close to each other and interact electrostatically in R5 viruses. This interaction was used to model V3 in the context of gp120 using NMR data for the V3 loop and the crystal structure of the gp120-core. The interaction between residues 322 and 440 may serve as part of the molecular switch for HIV-1 phenotype conversion.     

Identification of putative, stable binding regions through flexibility analysis of HIV-1 gp120

The acquired-immunodeficiency syndrome has evolved into a major worldwide epidemic. Significant effort has been made in the development of antiviral therapies. A new strategy for vaccine and drug design that complements the existing cocktail therapy is to target entry of the human immunodeficiency virus (HIV). Such an approach provides the advantage of interfering with multiple intermediates in this multi-step process. The extraordinary conformational flexibility, glycosylation, and strain variations of viral glycoprotein gp120 cause general viral evasion of humoral immune response and thus complicate the development of an effective vaccine. Especially difficult to define are the conformation of gp120 before CD4 engagement as well as the relative orientations of the V1/V2 and V3 loops with respect to the inner and outer domains. In this study, we used Floppy Inclusion and Rigid Substructure Topography (FIRST), a program based on graph theory, to analyze the flexibility and rigidity of all known HIV-1 gp120 structures. A flexibility index is used to describe and compare the spatial distribution of protein flexibility and rigidity of these structures in isolation and in complex with CD4, CD4-mimics, and neutralizing antibodies. Using this flexibility analysis, we identified a universal rigid region (the alpha2 helix) as well as the consensus largest rigid cluster involving a beta-sheet located on the coreceptor binding face. Both of these regions may serve as stable targets for vaccine design and drug discovery. Detailed comparisons of the changes in flexibility based on strain variations, stabilizing mutations, binding features of CD4 mimics, and impact of b12 binding are reported.     

Neuronal glycolytic pathway impairment induced by HIV envenlope glyocprotein gp120

Neurological impairment is a common feature of Acquired Immunodeficiency Syndrome (AIDS); functional alterations have been reported both in central and peripheral nervous system and the Human Immunodeficiency Virus (HIV) envelope glycoprotein gp120 has been proposed as a neurotoxin acting through a calcium-dependent mechanism. On the other hand it has been reported that gp120 treatment also induce about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels. The reported observations were performed on experimental system where also non-neuronal cells where present; in order to evaluate whether a direct interaction between HIV proteins and neuronal cells takes place, we used a neuroblastoma cultures where only neuronal cells are present.We analysed the effects of gp120 on the N18TG2 neuroblastoma clone. Treatments were performed both on growing and confluent cultures. Short time treatment with gp120 of confluent cultures causes a 25% reduction in the level of neuron-specific enolase, resulting in a similar decrease of oxygen consumption. Long time exposure of growing cells also causes a reduction in cell survival. Furthermore, using a membrane-specific fluorescent probe we observed that gp120 produces an increase of membrane trafficking. These observations suggest a direct interaction between the viral envelope protein and neuronal cells, which results in an alteration of glycolytic metabolism. This alteration may be related to the neurologic impairments observed in AIDS patients.     

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.     

The disulfide loop of gp41 is critical to the furin recognition site of HIV gp160

The importance of the HIV gp41 conserved disulfide loop to envelope function has been examined by mutational and functional analyses. Based on a luciferase-reporter entry assay, mutants gp41-CC/AA (C598A/C604A) and gp41-Delta (deletion of residues 596-606) result in a nonfunctional envelope protein. Western blot analysis shows both mutants to be properly expressed but not processed to form gp120 and gp41, which explains their nonfunctionality. The presence of mutant gp160 on the cell surface, as well as their ability to bind to sCD4, suggests that the mutations have disrupted processing at the furin recognition site encoded within the gp120 conserved domain 5, without resulting in an overall misfolding of the protein. With respect to the furin recognition site, the mutations are sequentially distant, which implies that the gp41 disulfide loop is interacting with gp120 C5 in gp160. In addition, we have modeled the gp120-gp41 interaction in unprocessed precursor gp160 using structural data available for gp120 and gp41 domains in isolation, supplemented by mutagenesis data. We suggest that the mutations have altered the interaction between gp120 C5 and the gp41 disulfide loop, resulting in decreased accessibility of the furin recognition site and implying that the interaction between the gp120 C5 and gp41 loop is a conformational requirement for gp160 processing. The sensitivity of this interaction could be exploited in future antivirals designed to disrupt HIV pathogenesis by disrupting gp160 processing.     

Modeling interaction between gp120 HIV protein and CCR5 receptor

The study of the process of HIV entry into the host cell and the creation of biomimetic nanosystems that are able to selectively bind viral particles and proteins is a high priority research area for the development of novel diagnostic tools and treatment of HIV infection. Recently, we described multilayer nanoparticles (nanotraps) with heparin surface and cationic peptides comprising the N‐terminal tail (Nt) and the second extracellular loop (ECL2) of CCR5 receptor, which could bind with high affinity some inflammatory chemokines, in particular, Rantes. Because of the similarity of the binding determinants in CCR5 structure, both for chemokines and gp120 HIV protein, here we expand this approach to the study of the interactions of these biomimetic nanosystems and their components with the peptide representing the V3 loop of the activated form of gp120. According to surface plasmon resonance results, a conformational rearrangement is involved in the process of V3 and CCR5 fragments binding. As in the case of Rantes, ECL2 peptide showed much higher affinity to V3 peptide than Nt (K_D = 3.72 × 10^?8 and 1.10 × 10^?6 M, respectively). Heparin‐covered nanoparticles bearing CCR5 peptides effectively bound V3 as well. The presence of both heparin and the peptides in the structure of the nanotraps was shown to be crucial for the interaction with the V3 loop. Thus, short cationic peptides ECL2 and Nt proved to be excellent candidates for the design of CCR5 receptor mimetics.     

The design of a specific ligand of HIV gp120

The crystal structure of CD4 suggested that the C/G³⁸ and C/L⁴⁴ replacements with the consequent cystine bridge formation are compatible with the native structure of that molecular moiety. As the NQGSF sequence, corresponding to the 39–43 fragment of human CD4 protein, was found to be involved in the HIV gp120 interaction, it has been synthesized in a cyclic form by adding two cysteine residues at the amino and carboxy termini. ¹H-NMR studies show that the predominant solution conformation of cyclo-[CNQGSFC] is a type II β-turn centred on the NQGS segment. Structural and dynamic properties of the peptide are also analysed in relation to the in vitro activity. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci. 3: 383–390 No. of Figures: 7. No. of Tables: 1. No. of References: 33.     

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

Background

HIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown.

A recombinant allosteric lectin antagonist of HIV-1 envelope gp120 interactions

The first, critical stage of HIV-1 infection is fusion of viral and host cellular membranes initiated by a viral envelope glycoprotein gp120. We evaluated the potential to form a chimeric protein entry inhibitor that combines the action of two gp120-targeting molecules, an allosteric peptide inhibitor 12p1 and a higher affinity carbohydrate-binding protein cyanovirin (CVN). In initial mixing experiments, we demonstrated that the inhibitors do not interfere with each other and instead show functional synergy in inhibiting viral cell infection. Based on this, we created a chimera, termed L5, with 12p1 fused to the C-terminal domain of CVN through a linker of five penta-peptide repeats. L5 revealed the same broad specificity as CVN for gp120 from a variety of clades and tropisms. By comparison to CVN, the L5 chimera exhibited substantially increased inhibition of gp120 binding to receptor CD4, coreceptor surrogate mAb 17b and gp120 antibody F105. These binding inhibition effects by the chimera reflected both the high affinity of the CVN domain and the allosteric action of the 12p1 domain. The results open up the possibility to form high potency chimeras, as well as noncovalent mixtures, as leads for HIV-1 envelope antagonism that can overcome potency limits and potential virus mutational resistance for either 12p1 or CVN alone.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

Disulfide Reduction in CD4 Domain 1 or 2 Is Essential for Interaction with HIV Glycoprotein 120 (gp120), which Impairs Thioredoxin-driven CD4 Dimerization

Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.     

重组HIV表面抗原gp120的表达纯化及免疫学鉴定

为研制具有流行特点的HIV血清学诊断试剂,采用pET系统表达HIV－1表达糖蛋白gp120。研究发现,全长的gp120在E.coli中不能有效表达;N端半长的gp120可以表达,但表达量很低;仅保留N端1／3的gp120（包含gp120V1／V2抗决定簇）有效表达,表达蛋白占菌体总蛋白的18％;Western blot显示较好的反应原性;通过金属螯合层析,产物得到完全纯化。在这些结果的基础上,我们表达了流行株的gp120片段,为探索gp120在大肠杆菌的高效表达,建立针对中国人群的HIV血清学诊断系统奠定基础。     

共同受体CCR5与HIV gp120的相互作用及相关肽类抑制剂

存在于巨嗜细胞、树突状细胞等胞膜上的G蛋白偶联受体CCR5作为R5嗜性的HIV-1病毒的主要共同受体,可以和病毒的表面糖蛋白gp120相互作用,并由此决定了病毒的另一表面糖蛋白gp41融合构象的形成以及随后的病毒与细胞的膜融合。CCR5在细胞膜上迅速移动,并与其他分子(如CD4和胆固醇)存在相互作用,加速了与gp120的作用。CCR5的这种中心作用已经使其成为抗HIV-1药物研究的很有吸引力的靶点。目前已发现一系列衍生于CCR5的胞外区的多肽、天然存在的蛋白质以及设计的多肽,可干扰CCR5与gp120之间的相互作用,从而抑制病毒复制。     

Conformational study of fragments of envelope proteins (gp120: 254-274 and gp41: 519-541) of HIV-1 by NMR and MD simulations.

The envelope proteins, gp 120 and gp41 of HIV-1, play a crucial role in receptor (CD4+ lymphocytes) binding and membrane fusion. The fragment 254-274 of gp120 is conserved in all strains of HIV and, as a part of the full gp120 protein, behaves as 'immunosilent', but as an individual fragment it is 'immunoreactive'. When this fragment binds to its receptor, it activates the fusion domain of gp41 allowing viral entry into the host CD4+ cells. The conformation of fragment 254-274 of the gp120 domain and fragment 519-541 of the gp41 domain was studied by NMR and MD simulations. The studies were carried out in three varied media--water, DMSO-d6 and hexafluoroacetone (HFA). The fusogenic nature of the gp41 domain peptide was investigated by 31P NMR experiments with model bilayers prepared from dimyristoyl-L-alpha-phosphatidylcholine (DMPC). The solvent was seen to exert a major effect on the structure of the two peptides. Fragment (254-274) of gp120 in DMSO-d6 had a type I beta-turn around the tetrad Val9-Ser10-Thr11-Gln12 while in HFA a helical structure spanning the region Ile5 to Gln12 was seen with the remaining part of the peptide in a random coil structure. It is possible that the beta-turn may constitute an initiation site for the formation of the helix. In water at pH 4.5, the peptide adopted a beta-sheet. The NMR results for fragment 519-541 of gp41 are conclusive of a beta-sheet structure in DMSO-d6, a conformation which may help in insertion into the membrane, a notion also put forward by others. The 31P NMR studies of DMPC vesicles with this fragment show its fusogenic nature, promoting fusion of unilamellar vesicles to larger agglomerates like multilamellar ones.     

人星形细胞HIV—1膜蛋白gp120新受体的免疫学鉴定

自1994年首次报导HIV－1外膜蛋白gp120与人胎儿星形细胞膜蛋白质位点（推测分子量为260kD,命名为PAG）结合以来[1],这项工作持续集中于研究该蛋白的功能与作用,本研究藉助杂交瘤技术建立了一系列不鼠抗人星形细胞PAG）的单克隆抗体,这些抗体成功的抑制了gp120与PAG的结合,抑制可达50％以上,并几乎完全阻断了gp120介导的由摄入所星表细胞风钙离子的升高,通过ELISA可证实抗体与星形细胞的特异反应,蛋白印迹和免疫沉淀试验结果表明PAG作为实体的存在,试验表明PAG对于gp120与星形细胞的结合,对于gp120个导的星形细胞摄入所致钙离子的升高均有决定性作用,许多方向报导和研究表明gp120可与多种细胞结合而导致HIV－1感染,由此推论PAG是HIV－1gp120在人星形细胞上的新受体,同时也可能对HIV－1脑病的治疗开辟一条崭新的途径,PAG是否为HIV－1感染人星形细胞的受体,仍有等进一步实验证明。     

Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (K_d = 1.59 nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide aptamers.     

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.