Anchor sequence-dependent endogenous processing of human immunodeficiency virus 1 envelope glycoprotein gp160 for CD4+ T cell recognition

Human CD4+ T cell clones and cell lines were shown to lyse recombinant vaccinia virus-infected cells that synthesize the HIV-1 envelope glycoprotein gp160. The processing of endogenously synthesized gp160 for recognition by CD4+ T cells required that the protein, after synthesis on the rough endoplasmic reticulum and during subsequent cellular transport, remain attached to the luminal/extracellular membrane face by a hydrophobic anchor sequence.     

Simple assay to screen for inhibitors of interaction between the human immunodeficiency virus envelope glycoprotein and its cellular receptor, CD4.

The binding of the human immunodeficiency virus envelope glycoprotein gp120 to the CD4 molecule is the initial step in the viral replicative cycle. This interaction is therefore an important target for therapeutic intervention for the treatment of human immunodeficiency virus infection. We designed an enzyme-linked immunosorbent assay which detects the interaction between recombinant soluble forms of CD4 and gp160. This assay could be used as an initial screen of libraries of synthetic chemical compounds and natural products.     

Role of N-linked glycans in the interaction between the envelope glycoprotein of human immunodeficiency virus and its CD4 cellular receptor. Structural enzymatic analysis

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.     

Neutralizing antibodies to human immunodeficiency virus type-1 gp120 induce envelope glycoprotein subunit dissociation

The spectrum of the anti-human immunodeficiency virus (HIV) neutralizing immune response has been analyzed by the production and characterization of monoclonal antibodies (mAbs) to the viral envelope glycoproteins, gp41 and gp120. Little is known, however, about the neutralization mechanism of these antibodies. Here we show that the binding of a group of neutralizing mAbs that react with regions of the gp120 molecule associated with and including the V2 and V3 loops, the C4 domain and supporting structures, induce the dissociation of gp120 from gp41 on cells infected with the T cell line-adapted HIV-1 molecular clone Hx10. Similar to soluble receptor-induced dissociation of gp120 from gp41, the antibody-induced dissociation is dose- and time- dependent. By contrast, mAbs binding to discontinuous epitopes overlapping the CD4 binding site do not induce gp120 dissociation, implying that mAb induced conformational changes in gp120 are epitope specific, and that HIV neutralization probably involves several mechanisms.     

Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.     

Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4

The human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1.     

Role of envelope glycoprotein carbohydrate in human immunodeficiency virus (HIV) infectivity and virus-induced cell fusion

Human immunodeficiency virus (HIV) envelope glycoprotein interactions with cell surface CD4 are involved in both virion infectivity and virally mediated cell fusion. D-mannose-specific lectins such as Con A specifically blocked virion infectivity and cell fusion. Studies with a recombinant vaccinia virus containing the HIV envelope gene demonstrated that Con A-mediated inhibition of HIV-induced fusion involved lectin binding to the viral envelope glycoprotein. These results indicate the importance of envelope glycosylation in the pathobiology of HIV infection, and suggest potential mechanisms for interfering with HIV infectivity and cytopathology.     

Elevated levels of soluble CD54 (ICAM-1) in human immunodeficiency virus infection

Soluble CD54 levels in sera were quantitated in asymptomatic intravenous drug users, homosexuals, and patients with lymphadenopathy, AIDS-related complex, or acquired immunodeficiency syndrome. Soluble CD54 levels were elevated in human immunodeficiency virus (HIV)-seronegative asymptomatic intravenous drug users, reflecting infections like cytomegalovirus, Epstein-Barr virus, and hepatitis B virus. The sera of human immunodeficiency virus-seropositive groups of patients also had elevated levels of soluble CD54, reflecting infections like cytomegalovirus and human immunodeficiency virus infection. © 1993 Wiley-Liss, Inc.     

The human immunodeficiency virus gp120 binding site on CD4: delineation by quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high-resolution CD4 atomic structure

The first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, Lys46, and Arg59, on the C, C', C", and D strands, respectively. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gp120. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction.     

Structure and function of oligosaccharide chains of the envelope glycoprotein gp120 of human immunodeficiency virus type 1]

The surface of the human immunodeficiency virus (HIV-1), a causative agent for acquired immunodeficiency syndrome (AIDS), is covered with the major envelope glycoprotein gp120, of which the carbohydrate moiety accounted for 50% of the molecular mass. There is evidence that glycosylation of gp120 is prerequisite to the various stages of HIV infection. The oligosaccharide structures of gp120 have been determined using recombinant gp120 of HIV-1 (IIIB) produced in chinese hamster ovary cells and virus-derived gp120 isolated from H9 lymphocytes chronically infected with HIV-1 (IIIB). Three oligosaccharides have been suggested to be involved in the HIV-infection process. Occurrence of infection process which is independent of CD4 recognition and mediated by gp120 oligosaccharides, mannose-binding protein, and complement system has been suggested.     

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity.     

Cells induced to express a human immunodeficiency virus type 1 envelope gene mutant inhibit the spread of wild-type virus.

The feasibility of using a trans-dominant interfering human immunodeficiency virus type 1 (HIV-1) envelope mutant for inducible gene therapy of HIV infection was investigated. Genes encoding wild-type or mutant glycoproteins were introduced into CD4+ cells, where they were stably maintained but not expressed until induced. Envelope (env) gene expression was dependent upon the viral regulatory protein Tat. Induction of the mutant env resulted in resistance to cytopathic effects mediated by wild-type envelope and decreased infectious vector virus production. When cells containing the mutant env gene were infected with wild-type virus, viral spread was inhibited. The fact that maintenance of the env gene was stable over time suggests that inducible gene therapy using the dominantly interfering env mutant may be a feasible approach to slowing the progression of HIV-1 disease.     

Infection of monocytes by human immunodeficiency virus type 1 blocked by inhibitors of CD4-gp120 binding, even in the presence of enhancing antibodies

Infection of monocyte/macrophages (M/M) by a variety of viruses (including HIV-1) has been shown to be enhanced in the presence of low concentrations of antiviral antibodies. This process has been hypothesized as occurring through binding of the virus-antibody complex to Fc or complement receptors followed by endocytosis. In the current study, we explored whether such a mechanism might provide a CD4-independent route of infection by HIV-1 for any of several populations of M/M. In the absence of anti-HIV antibodies, replication of HIV-1 in M/M was blocked by viral binding inhibitors such as soluble CD4 or OKT4A mAb. Furthermore, while infection of the M/M populations by a low multiplicity of infection of HIV-1 was found to be somewhat enhanced by the presence of very low concentrations of anti-HIV antibodies, this process was also consistently inhibited by recombinant soluble CD4 and by OKT4A antibody. These results suggest that under the variety of conditions studied, CD4 binding was an essential step in the infection of M/M by HIV. Moreover, they are consistent with the notion that "enhancing" antibodies may serve to concentrate HIV onto CD4 receptors or, alternately, may act at other steps in the process of viral entry and replication.     

CD4^＋T淋巴细胞在无症状人类免疫缺陷病毒感染者早期接受扶正排毒片治疗后的变化

背景:无症状人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)感染者不适宜抗病毒治疗,中医药早期干预对无症状带毒期HIV感染者会产生一定的治疗意义。目的:观察扶正排毒片早期干预HIV感染者CD4 T淋巴细胞水平及血浆病毒载体量变化。设计:观察对比试验。单位:河南中医学院艾滋病研究所。对象:于2005-11随机抽取HIV感染者较多的某乡69例有偿献血感染的无症状HIV感染者,男39例,女30例,年龄30～61岁,平均(43±8)岁,平均病程(13±3)年。纳入标准:①符合中华人民共和国国家艾滋病无症状HIV感染诊断标准(2005年修订版)。②年龄18～65岁。③200/mm3≤CD4 T淋巴细胞计数<600/mm3。④未使用抗病毒药物及其它药物治疗者。⑤签署知情同意书。排除标准:①严重肝肾功能不全,或合并心脑血管、肺和造血系统等严重原发性疾病,精神病患者。②原发性免疫缺陷患者,激素、化疗等引起的继发性免疫缺陷患者,血液病患者。③妊娠或哺乳期妇女。随机抽取的某个县的25例无症状HIV感染者为空白组,纳入标准、排除标准与治疗组相同。方法:治疗方案:治疗组感染者给予扶正排毒片(黄芪、生白术、防风、白花蛇舌草、甘草等11味,河南省中医研究院奥林特制药厂生产,产品批号:050601),每次5片,相当于生药6.6g,3次/d,温开水冲服。3个月为1疗程,共观察4个疗程。空白组不服药。主要观察指标:①检测治疗组感染者治疗前和每疗程结束后CD4 、CD8 及CD4 /CD8 值,空白组只检测6个月前后CD4 T淋巴细胞计数。②采用RT-PCR检测方法进行治疗组患者病毒载量检测。结果:治疗组感染者69例及空白组感染者25例均进入结果分析。①空白组感染者6个月前后CD4 较治疗前明显下降,差异有统计学意义(P<0.05)。②随机抽取23例治疗组感染者进行病毒载量检测,与疗前对比,疗后6个月有9例血浆病毒载量下降较多,疗后1年12例血浆病毒载量下降较多。疗前和疗后6个月及1年对比有效率均达91.30%。结论:扶正排毒片早期干预HIV感染者可提高其免疫功能,对部分病例具有降低病毒载量的作用。     

Additive effect of tunicamycin and dextran sulfate on the binding of monoclonal antibody to the V2 domain of the envelope glycoprotein 120 of human immunodeficiency virus type 1.

Using flow cytometry or immunoprecipitation analysis in cells chronically infected with HIV-1 IIIB Supt-1, we noticed an additive effect of tunicamycin and low molecular weight dextran (LMDS) on the binding of the G3-4 monoclonal antibody to monomeric and oligomeric forms of glycoprotein 120 (gp120). The inhibition of glycosylation by tunicamycin reduced the number of monomeric and oligomeric forms of gp120. The inhibition of the binding of the G3-4 antibody to monomeric and oligomeric forms of gp120 was more pronounced in the presence of LMDS. We also found that the G3-4 antibody can not recognise the nascent polypeptide chain of the envelope glycoprotein.     

Conferral of an antiviral state to CD4+ cells by a zipper motif envelope mutant of the human immunodeficiency virus type 1 transmembrane protein gp41.

We showed in a transient coexpression study that a single proline substitution for any of the five conserved leucine or isoleucine residues located in the envelope (Env) transmembrane protein gp41 zipper motif of the human immunodeficiency virus type 1 dominantly interferes with wild-type Env-mediated viral infectivity. In the present study, we intended to explore the feasibility of developing a genetic anti-HIV strategy targeting the zipper motif. Stable HeLa-CD4-LTR-beta-gal clones that harbored silent copies of Tat-regulated expression cassettes encoding the zipper motif Env mutants were first generated. Expression of any of the five Env mutants in transfectants interfered with exogenously expressed homologous HXB2 Env-mediated cytopathic effects. Mutant transfectants 566, 573, and 580 were further examined. Viral transmission mediated by the laboratory-adapted T cell-tropic HXB2 and NL4-3 viruses was greatly reduced in these transfectants compared with that observed in the env-defective control deltaKS and wt env transfectants. Moreover, viral replication mediated by the NL4-3 virus and a macrophage-tropic ADA-GG virus was delayed or reduced in human T cells harboring the mutant 566 or 580 env construct as opposed to those observed in cells harboring the control deltaKS or mutant 573 env construct. The wt and mutant Env proteins formed a hetero-oligomer when they were coexpressed. These results demonstrate that zipper motif Env mutants 566 and 580 confer an anti-HIV state to the host CD4+ cells, which indicates that dominant inhibitory mutants targeting the gp41 zipper motif might function as genetic anti-HIV agents to combat HIV-1 infection.     

Potent inhibition of human immunodeficiency virus type 1 replication by conditionally replicating human immunodeficiency virus-based lentiviral vectors expressing envelope antisense mRNA

We describe an HIV-based lentiviral vector that expresses a 1-kb antisense mRNA directed against the HIV-1 mRNAs containing env sequences. The expression of antisense env mRNAs (envAS) does not inhibit the synthesis of p24 expressed from the HIV-1 helper plasmid used to package the vector, as this helper has a deletion in the env gene. This allows the production of high-titer VSV-G pseudotyped lentiviral particles. In challenge experiments using unselected populations of SupT1 cells transduced with this vector, a complete inhibition of HIV-1 replication was observed for long periods of in vitro culture, even at high HIV-1 infectious doses. The potent inhibition of HIV-1 replication by this vector correlated with a low occurrence of mobilization of the vector to previously untransduced cells. The infectivity of the wild-type HIV-1 that escapes inhibition was highly inhibited, suggesting that the vector is providing HIV-1 inhibition of replication not only due to its antisense effect but also by competing for encapsidation and mobilization to noninfected cells.