The role of calcium andN-linked glycans in the oligomerization and carbohydrate binding properties of human immunodeficiency virus external envelope glycoprotein

Envelope glycoproteins of human immunodeficiency virus (gp120 and gp41) occur as oligomers. Here, we show by gel filtration analysis that gp 120 oligomerizationin vitro is calcium- and temperature-dependent. Recombinant gp120 (rgp120) species were recovered as monomers at 20 °C in the absence of calcium, but as tetramers at 37 °C in 10mm CaCl₂. Under the latter condition,N-glycanase-deglycosylated rgp120 formed hexamers. Relative to intact rgp120, which has been reported to display carbohydrate-binding properties forN-acetyl--d-glucosaminyl and mannosyl residues, deglycosylation enhanced rgp120 specific binding to mannose-divinylsulfone-agarose, para-aminophenyl--d-GlcNAc-agarose and fetuin-agarose matrices. Taken together, these results rule out the role of homologous lectin-carbohydrate interactions viaN-linked glycans in the rgp120 oligomerization, even though its lectin properties may also be calcium-dependent. Deglycosylation may unmask domains of rgp120 polypeptide backbone that independently play a role either in rgp120 lectin activity or in calcium-dependent oligomerization.     

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.     

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.     

Linear epitope mapping of humoral responses induced by vaccination with recombinant HIV-1 envelope protein gp160

To enhance utility of the linear epitope mapping (Pepscan) technique for assay of humoral responses linked to vaccination, two modifications were tested. First, peptides were incubated with serum contained in baths rather than individual wells. Second, a rigorous statistical model was developed to determine which peptide/antibody-binding interactions were significant. The modifications increased the ability to detect signal in these experiments by 15- to 45-fold. These two modifications were applied to linear epitope mapping of HIV seropositive volunteers under treatment with recombinant HIV gp160 and also to rabbits immunized with the same product. Changes in fine specificity of response were observed in animal models and human vaccine recipients over the course of an immunization series with this antigen. Received 16 July 1996/ Accepted in revised form 09 April 1997     

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.     

Triazine Dyes Inhibit HIV-1 Entry by Binding to Envelope Glycoproteins

We have attempted to purify envelope (Env) glycoproteins of human immunodeficiency virus (HIV) from the culture supernatants of CHO-Sec cells that secreted truncated 140-kDa precursor and mature 120-kDa Env glycoproteins. The concentrated culture supernatants were applied to a column coupled with cibacron blue 3GA (CB3GA) to separate albumin from the Env proteins because CB3GA, a triazine dye, has been known to have a high affinity to albumin. Unexpectedly, Env proteins as well as albumin bound to the column, and the bound Env proteins were eluted by increasing the ionic strength using KC1. Gp120 was eluted at 0.5–0.9 m of KC1, while a higher concentration (0.9–1.5 m ) was necessary for the elution of gp140. The agarose gel coupled with reactive red 120 (RR120), another triazine dye with similar characteristics, also retained both Env proteins, and the bound Env proteins could be eluted in a similar manner. In addition, these agents inhibited syncytium formation caused by HTLV-III_B and HTLV-II_MN. Inhibition was also seen when a virus-free fusion assay between Env protein expressed in CHO cells and fluorescent labeled SupT1 cells were used. These findings indicate that triazine dyes bind to the functional regions of Env proteins of HIV-1 that play important role(s) for HIV infection.     

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。     

Effects of signal peptide exchange on HIV-1 glycoprotein expression and viral infectivity in mammalian cells

In certain cell systems, exchange of the human immunodeficiency virus (HIV) Env signal peptide (SP) sequence with that of heterologous SPs has been shown to increase gp120 transport and secretion. Here we demonstrate that exchange of the HIV-Env-SP with those from erythropoietin or tissue plasminogen activator in the proviral context does not increase wild-type membrane-bound Env expression or incorporation into released virions. In fact, virion infectivity was decreased. These infectivity decreases were largely due to effects on Env transport and/or function and only to a minor extent to cis effects as a result of the sequence exchanges themselves. Thus, in fact, it is not advantageous to employ heterologous SPs to achieve high-level expression of functional cell surface membrane- or virion-associated HIV-Env.     

Characterization of monoclonal antibodies to the envelope proteins of an immunodeficiency virus of African green monkeys,SIVagmTYO-7

Five monoclonal antibodies (mabs) specific for the envelope proteins of a simian immunodeficiency virus of African green monkeys (SIV_agm) have been raised. Two mabs were directed against distinct epitopes on the transmembrane protein gp41. A conformational epitope on the gp130 was recognized by three mabs. This is the first report on mabs specific for SIV_agm-gp130. Studies of the cross-reactivities revealed that the epitopes recognized by the env-directed mabs are conserved species-specifically in SIV_agm isolates. Therefore, these mabs can be used to distinguish SIV_agm strains from other virus groups.     

Heterogeneity of envelope molecules shown by different sensitivities to anti-V3 neutralizing antibody and CXCR4 antagonist regulates the formation of multiple-site binding of HIV-1

Increased temperature enhances the infectivity of human immunodeficiency virus type 1 (HIV-1), and this enhancement is inhibited by anti-CXCR4 peptide T140, implying that multiple-site binding is required to proceed to infection. Here, we tested whether the augmented infectivity induced by increased temperature could account for the heterogeneity of envelope molecules in the effectiveness of anti-V3 neutralization and anti-CXCR4 blocking. Pseudoviruses with the X4 envelope which were infectious at room temperature (RT) were more resistant to both anti-V3 neutralizing antibody 0.5beta and T140 than viruses infectious at 37 C and 40 C. Viruses infectious to cells treated with T140 were also resistant to 0.5beta. Based on the hypothesis that the HIV-1 viruses were carrying heterogeneity of functional and nonfunctional gp120 and required the formation of sufficient multiple-site binding of functional gp120 with receptors to proceed to infection, viruses with many functional gp120 which were infectious at RT and infectious to cells with reduced numbers of CXCR4 by T140 treatment were resistant to 0.5beta. Although viruses with many functional gp120 are a minority (less than 5%) of the infectious HIV-1 fraction, they are regarded as able to escape from neutralizing antibodies and coreceptor antagonists.     

Structural mimicry of CD4 by a cross-reactive HIV-1 neutralizing antibody with CDR-H2 and H3 containing unique motifs

Human immunodeficiency virus (HIV) entry into cells is initiated by the binding of its envelope glycoprotein (Env) gp120 to receptor CD4. Antibodies that bind to epitopes overlapping the CD4-binding site (CD4bs) on gp120 can prevent HIV entry by competing with cell-associated CD4; their ability to outcompete CD4 is a major determinant of their neutralizing potency and is proportional to their avidity. The breadth of neutralization and the likelihood of the emergence of antibody-resistant virus are critically dependent on the structure of their epitopes. Because CD4bs is highly conserved, it is reasonable to hypothesize that antibodies closely mimicking CD4 could exhibit relatively broad cross-reactivity and a high probability of preventing the emergence of resistant viruses. Previously, in a search for antibodies that mimic CD4 or the co-receptor, we identified and characterized a broadly cross-reactive HIV-neutralizing CD4bs human monoclonal antibody (hmAb), m18. Here, we describe the crystal structure of Fab m18 at 2.03 A resolution, which reveals unique conformations of heavy chain complementarity-determining regions (CDRs) 2 and 3 (H2 and H3). H2 is highly bulged and lacks cross-linking interstrand hydrogen bonds observed in all four canonical structures. H3 is 17.5 A long and rigid, forming an extended beta-sheet decorated with an alpha-turn motif bearing a phenylalanine-isoleucine fork at the apex. It shows striking similarity to the Ig CDR2-like C'C' region of the CD4 first domain D1 that dominates the binding of CD4 to gp120. Docking simulations suggest significant similarity between the m18 epitope and the CD4bs on gp120. Fab m18 does not enhance binding of CD4-induced (CD4i) antibodies, nor does it induce CD4-independent fusion mediated by the HIV Env. Thus, vaccine immunogens based on the m18 epitope structure are unlikely to elicit antibodies that could enhance infection. The structure can also serve as a basis for the design of novel, highly efficient inhibitors of HIV entry.     

中国HIV-1流行毒株的DNA疫苗的初步研究

为研制针对我国ＨＩＶ－１流行毒株的艾滋病毒疫苗,构建了具有代表性的ｇａｇ和ｇｐ１２０核酸疫苗,进行了初步的小鼠免疫实验。结果初步显示：（１）免疫Ｂａｌｂ／Ｃ小鼠可以产生ＨＩＶ－１特异性的体液和细胞免疫;（２）ｇａｇ和ｇｐ１２０基因联合免疫可以同时诱发针对ｇａｇ和ｇｐ１２０的细胞和体液免疫反应,而且效果比各自单独免疫要好;（３）Ｂ亚型ｇｐ１２０基因免疫可以诱发识别Ｃ亚型ｇｐ１２０抗原的ＣＴＬ反应。本     

Structure-function relationship of the mammarenavirus envelope glycoprotein

Mammarenaviruses, including lethal pathogens such as Lassa virus and Junín virus, can cause severe hemorrhagic fever in humans. Entry is a key step for virus infection, which starts with binding of the envelope glycoprotein(GP) to receptors on target cells and subsequent fusion of the virus with target cell membranes. The GP precursor is synthesized as a polypeptide, and maturation occurs by two cleavage events, yielding a tripartite GP complex(GPC) formed by a stable signal peptide(SSP), GP1 and GP2. The unique retained SSP interacts with GP2 and plays essential roles in virion maturation and infectivity. GP1 is responsible for binding to the cell receptor, and GP2 is a class I fusion protein. The native structure of the tripartite GPC is unknown.GPC is critical for the receptor binding, membrane fusion and neutralization antibody recognition.Elucidating the molecular mechanisms underlining the structure–function relationship of the three subunits is the key for understanding their function and can facilitate novel avenues for combating virus infections. This review summarizes the basic aspects and recent research of the structure–function relationship of the three subunits. We discuss the structural basis of the receptor-binding domain in GP1, the interaction between SSP and GP2 and its role in virion maturation and membrane fusion, as well as the mechanism by which glycosylation stabilizes the GPC structure and facilitates immune evasion. Understanding the molecular mechanisms involved in these aspects will contribute to the development of novel vaccines and treatment strategies against mammarenaviruses infection.     

Detection of IgA-binding sites on human immunodeficiency virus type-1 envelope glycoproteins, Gp120 and Gp41

IgA has been supposed to play an important role in the prevention of HIV-1 infection. In this study, IgA-binding sites on gp120 and gp41 of HIV-1 envelope glycoproteins were analyzed using ELISA and overlapping synthetic peptides covering all of the gp120 and gp41 sites. IgA antibodies in plasma and saliva mainly bound to six and five sites on gp120 and gp41, respectively. Some of the IgA-binding sites differed from those of IgG-binding sites and the amount of IgA antibodies that bound to each site varied among samples. IgA antibodies in some plasma samples neutralized HIV-1 infection, and those IgA antibodies contained the antibodies which bound to the V3, C3 and ELDKWA sites. The results suggest that IgA antibodies which bind to certain sites on HIV-1 envelope glycoproteins may neutralize HIV-1 infection, presumably at mucosal sites where most IgA antibodies are produced. The induction of IgA antibodies that bind specific sites and neutralize HIV-1 infection at mucosal sites may be important in the development of a vaccine against HIV-1 infection.     

HIV-1 envelope accessible surface and polarity: clade, blood, and brain

The human immunodeficiency virus type-1 (HIV-1) gp160 (gp120-gp41 complex) trimer envelope (ENV) protein is a potential vaccine candidate for HIV/AIDS. HIV-1 vaccine development has been problematic and charge polarity as well as sequence variation across clades may relate to the difficulties. Further obstacles are caused by sequence variation between blood and brain-derived sequences, since the brain is a separate compartment for HIV-1 infection. We utilize a threedimensional residue measure of solvent exposure, accessible surface area (ASA), which shows that major segments of gp120 and gp41 known structures are solvent exposed across clades. We demonstrate a large percent sequence polarity for solvent exposed residues in gp120 and gp41. The range of sequence polarity varies across clades, blood, and brain from different geographical locations. Regression analysis shows that blood and brain gp120 and gp41 percent sequence polarity range correlate with mean Shannon entropy. These results point to the use of protein modifications to enhance HIV-1 ENV vaccines across multiple clades, blood, and brain. It should be noted that we do not address the issue of protein glycosylation here; however, this is an important issue for vaccine design and development. ABBREVIATIONS: HIV-1 - human immunodeficiency virus type 1, AIDS - acquired immunodeficiency syndrome, ENV - envelope, gp160 - 160,000d glycoprotein, gp120 - 120,000d glycoprotein, gp41 - 41,000d glycoprotein, LANL - Los Alamos National Laboratories, PDB - Protein Data Bank, HVTN - STEP HIV vaccine trial, AA - amino acids, MSA - multiple sequence alignment, ASA - accessible surface area, SNPs- single nucleotide polymorphisms, HAART - Highly Active Antiretroviral Therapy, CCR5 - C-C chemokine receptor type 5, CNS - central nervous system, HIVE - HIV encephalitis, P - polarity, NP - non-polarity, CTL - cytotoxic T lymphocyte, NIAID - National Institute of Allergy and Infectious Diseases.     

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.     

生物质谱技术在糖蛋白结构分析中的应用

生物质谱包括基质辅助激光解吸附飞行时间质谱及电喷雾质谱被广泛应用于生物样品如多肽、蛋白质及核酸的分析,由于这种具有软电离方式的质谱具有极高的灵敏度及准确度,目前也被成功地用于糖蛋白的结构分析,与普通的化学方法相比,质谱法快速、简单,结合网上数据库检索、凝集素亲和提取、二维凝胶电泳以及靶上直接酶切等新方法,可以提供糖蛋白的一级结构乃至高级结构的信息。     

Enhanced anandamide degradation is associated with neuronal apoptosis induced by the HIV-1 coat glycoprotein gp120 in the rat neocortex

Human immunodeficiency virus type-1 coat glycoprotein gp120 causes delayed apoptosis in rat brain neocortex. Here, we investigated the possible role of the endocannabinoid system in this process. It is shown that gp120 causes a time-dependent increase in the activity and immunoreactivity of the anandamide (AEA)-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), paralleled by increased activity of the AEA membrane transporter and decreased endogenous levels of AEA. The AEA-synthesizing phospholipase D and the AEA-binding receptors were not affected by gp120. None of the changes induced by gp120 in the cortex were induced by bovine serum albumin, nor were they observed in the hippocampus of the same animals. Also, the activity of 5-lipoxygenase, which generates AEA derivatives able to inhibit FAAH, decreased down to approximately 25% of the control activity upon gp120 treatment, due to reduced protein level ( approximately 45%). In addition, the FAAH inhibitor methyl-arachidonoyl fluorophosphonate significantly reduced gp120-induced apoptosis in rat brain neocortex, whereas selective blockers of AEA membrane transporter or of AEA-binding receptors were ineffective. Taken together, these results suggest that gp120, by activating FAAH, decreases endogenous levels of AEA, and the latter effect seems instrumental in the execution of delayed neuronal apoptosis in the brain neocortex of rats.     

HIV-1跨膜蛋白gp41螺旋结构的原核表达及功能研究

HIV-1跨膜蛋白gp41是HIV-1包膜与靶细胞膜的融合过程中的关键蛋白,是理想的HIV-1融合抑制剂靶点。为开展以gp41为靶点的抑制剂筛选,以HIV-1 B亚型病毒基因为模板,通过PCR、酶切、连接等方法构建得到gp41 5-helix与6-helix重组质粒,转入大肠杆菌BL21（DE3）进行表达,经变性和复性后亲和层析纯化蛋白。经SDS-PAGE鉴定,纯化后蛋白纯度较高。本研究还通过非变性凝胶电泳证明gp41 5-helix与C-多肽衍生物T-20存在相互作用,为下一步药物筛选模型的建立奠定了基础。