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.     

HIV/AIDS患者CD4水平对甲状腺功能影响的比较研究

目的观察HIV/AIDS患者甲状腺功能的变化,了解HIV对内分泌的影响。方法检测168例HIV/AIDS患者TT4、TT3、FT4、FT3、TSH,并分析不同CD4水平对甲状腺功能影响。结果HIV/AIDS患者甲状腺功能可保持正常,但低CD4组的FT3、TSH水平低于高CD4组。结论HIV/AIDS患者甲状腺功能可保持正常,但HIV感染不同阶段会对内分泌系统有一定影响。HIV感染对甲状腺影响的具体机制尚不十分清楚,利用动物模型进行进一步研究非常必要。     

CD4+CD25+FoxP3+ 调节性T 细胞与黑龙江省HIV/AIDS 患者病情 相关性的研究

目的:比较黑龙江省HIV/AIDS患者与健康对照者(healthy controls,HCs)外周血CD4+CD25+FoxP3+调节性T细胞数量、免疫抑制功能的变化,探讨CD4+CD25+FoxP3+调节性T细胞在HIV/AIDS感染过程中的作用。方法:采用流式细胞仪检测21例HIV/AIDS患者及20例健康对照组的外周血CD4+CD25+FoxP3+调节性T细胞数量的百分比及绝对数量;采用共同培养方法检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞免疫抑制功能的变化;实时荧光定量聚合酶链反应(RT-FQ-PCR)检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞中FoxP3mRNA的表达。结果:黑龙江省HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞比率明显高于HCs(P<0.01),而CD4+CD25+FoxP3+调节性T细胞的绝对计数显著下降,且与CD4+T细胞绝对计数成反比;混合淋巴细胞共同培养结果显示,HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的抑制功能无明显变化;HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的FoxP3 mRNA相对表达量无显著变化。结论:黑龙江省HIV/AIDS患者CD4+CD25+FoxP3+调节性T细胞的数量变化与病情相关。     

A CD4 mimic as an HIV entry inhibitor: Pharmacokinetics

To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies.     

HIV-1 envelope glycoproteins-mediated apoptosis is regulated by CD4 dependent and independent mechanisms

The progressive loss of CD4 T lymphocytes is one of the hallmarks of HIV infection. The reverse correlation observed in vivo, between plasmatic HIV levels and CD4 T lymphocyte counts, supports the concept that direct HIV-mediated cell death contributes to this depletion. Previously, we and others have demonstrated, in vitro, that interactions between membrane-expressed HIV-envelope glycoprotein complexes and CD4 ecto-molecules are critical to cell killing which occurs mainly by apoptosis. Here, by the use of a co-culture model, in which chronically HIV-1 infected cells trigger apoptosis in uninfected CD4+ target cells, we have investigated the role of different CD4 domains in HIV envelope-mediated apoptosis. Target cells were A201 lymphoblastoid cell lines expressing wild-type CD4 or mutant forms of CD4. We show that the cytoplasmic domain of CD4 was not required for apoptosis induction. In contrast, the HIV permissive cell line expressing a CD4/CD8 chimeric molecule which contains only the first 171 amino acids of CD4, appeared to be resistant to HIV-induced apoptosis; thus suggesting that the D3-D4 CD4 module plays somewhat a regulatory role. Pre-treatment of wild-type CD4 expressing target cells by the phorbol ester PMA which leads to down-regulation of CD4, completely abolished apoptosis. Interestingly, in cells expressing CD4 devoid of its cytoplasmic domain, PMA blocked partially cell death without affecting, as expected, the CD4 expression. Taken together, these results demonstrate that although CD4 expression is essential for HIV envelope induced apoptosis, the apoptotic signal could be delivered in the absence of its cytoplasmic domain. Consistent with this, we suggest that other membrane associated molecule(s) are recruited for the signalling to initiate apoptosis.     

The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells

In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4⁺ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4⁺ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56^Lck, indicating that Siva-1 participates in a pathway independent of the p56^Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

HIV may produce inhibitory microRNAs (miRNAs) that block production of CD28, CD4 and some interleukins

It is well-known that HIV-1 infection results in a gradual decline of the CD4+ T-lymphocytes, but the underlying mechanism of this decline is not completely understood. Research has shown that HIV-1 infection of CD4+ T cells results in decreased CD28 expression, but the mechanism of this repression is unknown. There is also substantial evidence demonstrating regulatory involvement of microRNA (miRNA) during protein expression in plants and some animals, and reports have recently been published confirming the existence of viral-encoded miRNAs. Based on these findings, we hypothesize that viral-encoded miRNA from HIV-1 may directly alter T cell, macrophage and dendritic cell activity. To investigate a potential correlation between the genomic complementarity of HIV-1 and host cell protein expression, a local alignment search was performed to assess for regions of complementarity between the HIV-1 proviral genome and the mRNA coding sequence of various proteins expressed by CD+ T cells and macrophages. Regions of complementarity with strong correlations to the currently established criteria for miRNA:target mRNA activity were found between HIV-1 and CD28, CTLA-4 and some interleukins, suggesting that HIV-1 may produce translational repression in host cells.     

CD32+CD4+ T Cells Are Highly Enriched for HIV DNA and Can Support Transcriptional Latency

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Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.     

Functional impairment of CD4 T cells despite normalization of T cell number in HIV

Using an in vitro model, we demonstrate that when CD4 T cells from HIV infected subjects are enriched from total blood lymphocytes the immune response to antigen is augmented. However, augmentation of this response is confined to HIV infected subjects with relatively preserved CD4 T cell counts. Enriching for CD4 T cells had no effect on antigen responses in patients with low CD4 lymphocyte counts. These findings support the concept that CD4 T cells in late stage HIV have inherent qualitative defects.     

SMARThivCD4mos: a complexity-free and cost effective model technology for monitoring HIV patients CD4 number in resource-poor settings

We design a "simple" and "low cost" model technology for monitoring HIV patients CD4 number in resource-poor settings: SMARThivCD4mos. Cost and complexity are the major challenges to the developing world in transferring and implementing HIV surveillance technologies. We previously proposed a "three tests" combo kit model for improving HIV patients monitoring standards in resource-poor settings. From the pool of recommended alternative CD4 counting technologies, our "three tests" combo kit model retained the Capcellia technology for its "simplicity". However, compared to other CD4 counting technologies, such as Dynabeads, the Capcellia technology is "expensive". Here, we describe a cost reduction strategy of the Capcellia technology.     

Mathematical analysis of the global dynamics of a model for HIV infection of CD4+ T cells

A mathematical model that describes HIV infection of CD4(+) T cells is analyzed. Global dynamics of the model is rigorously established. We prove that, if the basic reproduction number R(0) < or = 1, the HIV infection is cleared from the T-cell population; if R(0) > 1, the HIV infection persists. For an open set of parameter values, the chronic-infection equilibrium P* can be unstable and periodic solutions may exist. We establish parameter regions for which P* is globally stable.     

Profiling of the CD4 receptor complex proteins

Intermolecular complexes produced by the CD4 molecule were studied. To preserve the integrity of weak protein-protein interactions of the CD4 antigen, cells were lysed in a mild nonionic detergent Brij97. Protein constituents of the complex were identified by our previously proposed fluorescence immunoprecipitation assay with subsequent mass spectrometry. In total, 26 proteins associated with CD4 were identified on CEM cells. The CD4 complex included the following major components: tyrosine phosphatase CD45, transferrin receptor CD71, tyrosine kinase Lck, and a lymphocyte phosphatase-associated phosphoprotein LPAP. The CD4 complex also contained some components of cytoskeleton and heat shock proteins. The association between CD4, CD71, and CD45 molecules was confirmed by immunoblotting. The CD4 complexes were not detected on the U937 myeloid cells lacking Lck and LPAP. We attempted to quantitatively characterize the CD4 complex composition.     

HIV-specific CD8+ T cell responses to HXB2 Gag and Nef peptide pools in Chinese HIV/AIDS patients

HXB2 is primarily used as a template strain in developing HIV vaccines in Europe and the US. However,it is not yet known whether the strain can induce strong HIV-specific CD8+ T cell responses in Chinese HIV/AIDS patients. In the present study,two groups of subjects were investigated:9 AIDS patients and 7 long-term nonprogressors (LTNPs). HIV-specific CD8+ T cell responses were examined in all patients through the ELISPOT assay. CD4+ T cell counts,CD8+ T cell counts,viral load and HIV subtype of each patien...     

CD45 variant alleles: possibly increased frequency of a novel exon 4 CD45 polymorphism in HIV seropositive Ugandans

The CD45 (leucocyte common) antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor signalling in lymphocytes, and expression of different CD45 isoforms is associated with distinct functions. Here we describe a novel polymorphism in exon 4 (A54G) of the gene encoding CD45 (PTPRC) that results in an amino acid substitution of Thr-19 to Ala in exon 4. The 54G allele was identified in African Ugandan populations and was found with a suggestive but not statistically significant increase in frequency amongst HIV-seropositive Ugandans. This suggests that the 54G variant and CD45 splicing abnormalities might be associated with HIV infection.     

CD4+ T-cell gene expression of healthy donors,HIV-1 and elite controllers: Immunological chaos

ObjectivesT-cell repertoire dysfunction characterizes human immunodeficiency virus type 1 (HIV-1) infection, but the pathogenic mechanisms remain unclear. Disease progression is probably due to a profound dysregulation of Th1, Th2, Th17 and Treg patterns. The aim of this study was to analyze the features of CD4+ T cells in HIV-positive patients with different viroimmunological profile.Methodswe used a gene expression dataset of CD4+ T cells from healthy donors, HIV+ naive patients and Elite Controllers (EC), obtained from the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE18233).ResultsPrincipal Component Analysis (PCA) showed an almost complete overlap between the HIV-infected and EC patients, which cannot easily explain the different responses to HIV infection of these two group of patients. We have found that HIV patients and the EC showed an upregulation of the Th1 pro-inflammatory cytokines and chemokines, compared to the controls. Also, we have surprisingly identified IL28B, which resulted downregulated in HIV and EC compared to healthy controls. We focused attention also on genes involved in the constitution of the immunological synapse and we showed that HLA class I and II genes resulted significantly upregulated in HIV and in EC compared to the control. In addition to it, we have found the upregulation of others syncytial molecules, including LAG3, CTLA4, CD28 and CD3, assisting the formation of syncytia with APC cells.ConclusionsUnderstanding the mechanisms of HIV-associated immunological chaos is critical to strategically plan focused interventions.     

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.     

HIV Nef is Secreted in Exosomes and Triggers Apoptosis in Bystander CD4+ T Cells

The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879–887). Extracellular Nef protein has deleterious effects on CD4⁺ T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4⁺ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099–3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302–310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro . Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4⁺ T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).