The anti-HIV pentameric pseudopeptide HB-19 is preferentially taken up in vivo by lymphoid organs where it forms a complex with nucleolin.

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of HIV infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. It blocks virus-particle attachment to permissive cells by binding and forming a stable complex with nucleolin expressed on the cell surface. Here, we have investigated the tissue distribution of the tritiated HB-19 by using beta-radio imager whole-body mapping in rats. A rapid, selective, and stable distribution and accumulation of the systematically administered HB-19 was demonstrated within the spleen, liver, bone, and kidney as soon as 5 min following its administration. No apparent uptake of HB-19 occurred in the brain and the muscle tissue. Interestingly and despite its rapid clearance from the blood, at 24 h postexposure a significant proportion of HB-19 was still recovered from target organs, of which 16-37% could be accounted for intact pseudopeptide. The elimination of HB-19 mainly occurred by renal glomerular filtration and most of the excreted radioactivity appeared to be HB-19 metabolites. Finally, injection of the biotin-labeled HB-19 pseudopeptide but not its control counterpart allowed the recovery of the HB-19-nucleolin complex from the liver, spleen, thymus, and bone marrow, thus indicating that the in vivo molecular target of HB-19 is surface nucleolin. Our results demonstrate the preferential uptake and stability of HB-19 in lymphoid organs that are the site of HIV propagation.

Inhibition of HIV infection by the cytokine midkine.

The growth factor midkine (MK) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles. Here we show that synthetic and recombinant preparations of MK inhibit in a dose-dependent manner infection of cells by T-lymphocyte- and macrophage-tropic HIV-1 isolates. The binding of labeled MK to cells is prevented by excess unlabeled MK or by the anti-HIV pseudopeptide HB-19 that blocks HIV entry by forming a stable complex with the cell-surface-expressed nucleolin. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-gamma and activation of T-lymphocytes by PHA or antibodies specific to CD3/CD28. In MK-producing lymphocytes, MK is detectable at the cell surface where it colocalizes with the surface-expressed nucleolin. Finally, by using MK-producing CD4(+) and CD4(-) cell clones we show that HIV infection in cell cultures could be inhibited in both an autocrine and a paracrine manner. The potent and distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli suggests that MK is a cytokine that could be implicated in HIV-induced pathogenesis.

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton.

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. The cell-surface-expressed nucleolin becomes clustered at the external side of the plasma membrane when cross-linked by the nucleolin-specific monoclonal antibody mAb D3. This clustering, occurring at 20 degrees C and in a well-organized pattern, is dependent on the existence of an intact actin cytoskeleton. At 37 degrees C, mAb D3 becomes internalized, thus illustrating that surface nucleolin can mediate intracellular import of specific ligands. Our results point out that nucleolin should also be considered a component of the cell surface where it could be functional as a cell surface receptor for various ligands reported before.

Characterization of the gene encoding the 100-kDa form of human 2',5' oligoadenylate synthetase.

The 2'-5' oligoadenylate synthetases (OAS) represent a family of interferon (IFN)-induced proteins implicated in the antiviral action of IFN. When activated by double-stranded (ds) RNA, these proteins polymerize ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n greater than or = 1. Three forms of human OAS have been described corresponding to proteins of 40/46, 69/71, and 100 kDa. These isoforms are encoded by three distinct genes clustered on chromosome 12 and exhibit differential constitutive and IFN-inducible expression. Here we describe the structural and functional analysis of the gene encoding the large form of human OAS. This gene has 16 exons with exon/intron boundaries that are conserved among the different isoforms of the human OAS family, reflecting the evolutionary link among them. The promoter region of the p100 gene is composed of multiple features conferring direct inducibility not only by IFNs but also by TNF and all-trans retinoic acid. In contrast, the induction of the p100 promoter by dsRNA is indirect and requires IFN type I production.

The HIV-1 gp120 inhibits the binding of adenosine deaminase to CD26 by a mechanism modulated by CD4 and CXCR4 expression.

HIV-1 external envelope glycoprotein gp120 inhibits adenosine deaminase (ADA) binding to its cell surface receptor in lymphocytes, CD26, by a mechanism that does not require the gp120-CD4 interaction. To further characterize this mechanism, we studied ADA binding to murine clones stably expressing human CD26 and/or human CD4, and transiently expressing human CXCR4. In this heterologous model, we show that both recombinant gp120 and viral particles from the X4 HIV-1 isolate IIIB inhibited the binding of ADA to wild-type or catalytically inactive forms of CD26. In cells lacking human CXCR4 expression, this gp120-mediated inhibition of ADA binding to human CD26 was completely dependent on the expression of human CD4. In contrast, when cells were transfected with human CXCR4 the inhibitory effect of gp120 was significantly enhanced and was not blocked by anti-CD4 antibodies. These data suggest that the interaction of gp120 with CD4 or CXCR4 is required for efficient inhibition of ADA binding to CD26, although in the presence of CXCR4 the interaction of gp120 with CD4 may be dispensable.

PKR stimulates NF-kappaB irrespective of its kinase function by interacting with the IkappaB kinase complex.

The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-kappaB. NF-kappaB is retained in the cytoplasm through binding to its inhibitor IkappaBalpha. The critical step in NF-kappaB activation is the phosphorylation of IkappaBalpha by the IkappaB kinase (IKK) complex. This activity releases NF-kappaB from IkappaBalpha and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-kappaB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2alpha-binding domain, can also activate NF-kappaB. This result indicated that NF-kappaB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2alpha relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR(0/0) mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathione S-transferase pull-down assay, we showed that PKR interacts with the IKKbeta subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-kappaB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-kappaB signaling pathway without using its kinase activity.

The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP inhibits attachment of T lymophocyte- and macrophage-tropic HIV to permissive cells.

The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP[psi(CH2N) indicating a reduced peptide bond], which binds the cell surface-expressed nucleolin, is a potent inhibitor of HIV infection. Here, by using primary T lymphocyte cultures and an experimental cell model to monitor HIV entry, we show that HB-19 inhibits in a dose-dependent manner both T lymphocyte- and macrophage-tropic HIV isolates. Similar positively charged control pseudopeptides have no effect on HIV infection even at high concentrations. These observations, and the fact that HB-19 has no effect on SIV-mac and HIV-1 pseudotyped with VSV envelope glycoproteins, confirm the specific nature of this inhibitor against the entry process mediated by the HIV envelope glycoproteins. Finally, association of low doses of HB-19 with beta-chemokines or AZT results in an increased inhibitory effect on HIV infection. HB-19 has no inhibitory effect when added to cells a few hours after HIV entry. On the other hand, in HB-19-pretreated cells, the inhibitory effect persists for several hours, even after washing cells to remove away the unbound pseudopeptide. Under such conditions, the attachment of HIV particles to cells is inhibited as efficiently as by neutralizing monoclonal antibodies directed against the V3 loop. In view of its specific mode of action on various HIV isolates, HB-19 represents a potential anti-HIV drug.

The anti-HIV pseudopeptide HB-19 forms a complex with the cell-surface-expressed nucleolin independent of heparan sulfate proteoglycans.

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of human immunodeficiency virus (HIV) infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. Here, by using an experimental CD4(+) cell model to monitor HIV entry and infection, we demonstrate that HB-19 binds the cell surface and inhibits attachment of HIV particles to permissive cells. At concentrations that inhibit HIV attachment, HB-19 binds cells irreversibly, becomes complexed with the cell-surface-expressed nucleolin, and eventually results in its degradation. Accordingly, by confocal immunofluorescence microscopy, we demonstrate the drastic reduction of the cell-surface-expressed nucleolin following treatment of cells with HB-19. HIV particles can prevent the binding of HB-19 to cells and inhibit complex formation with nucleolin. Such a competition between viral particles and HB-19 is consistent with the implication of nucleolin in the process of HIV attachment to target cells. We show that another inhibitor of HIV infection, the fibroblast growth factor-2 (FGF-2) that uses cell-surface-expressed heparan sulfate proteoglycans as low affinity receptors, binds cells and blocks attachment of HIV to permissive cells. FGF-2 does not prevent the binding of HB-19 to cells and to nucleolin, and similarly HB-19 has no apparent effect on the binding of FGF-2 to the cell surface. The lack of competition between these two anti-HIV agents rules out the potential involvement of heparan sulfate proteoglycans in the mechanism of anti-HIV effect of HB-19, thus pointing out that nucleolin is its main target.

Colocalization within the nucleolus of two highly related IFN-induced human nuclear phosphoproteins with nucleolin.

We have previously reported the identification of two interferon (IFN)-induced cDNAs which code for two proteins, named 41 and 75, which have homology to a number of proteins involved in regulating gene expression. Here we establish that these cDNAs correspond to in vivo synthesized mRNAs. Expression of the 41 and 75 cDNAs, both in vitro and in vivo, generated proteins of 30 and 68 kDa, respectively. In a variety of mammalian cells, 41 and 75 were found to be located in the nucleus, with 41 being localized to the nucleolus, whereas 75, although it is mainly concentrated at the periphery of the nucleolus, is also found throughout the nucleoplasm. Treatment with interferon results in a translocation of 41 to the periphery of the nucleolus and it is in this region that the two proteins colocalize. 41 and 75 were found to colocalize with nucleolin but not with B23 or fibrillarin, three nucleolar proteins involved in ribosome synthesis. This colocalization was not affected by low concentrations of actinomycin D. In view of this and since 41 and 75 have homology to proteins involved in regulating gene expression, we suggest that, in association with nucleolin, they play a role in ribosome biogenesis.

The human 2',5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties.

2',5'-Oligoadenylate synthetase (2',5'-OAS) was discovered and characterized as an interferon (IFN)-induced enzyme that in the presence of double-stranded (ds) RNA converts ATP into 2',5'-linked oligomers of adenosine with the general formula pppA(2'p'A)n, n > or = 1. The product is pppG2'p5'G when GTP is used as a substrate. Now, 20 years later, this activity is attributed to several well-characterized, homologous, and IFN-induced proteins in human cells. Three distinct forms of 2',5'-OAS exist, small, medium, and large, which contain 1, 2, and 3 OAS units, respectively, and are encoded by distinct genes clustered on the 2',5'-OAS locus on human chromosome 12. Recently, other IFN-induced proteins homologous to the OAS unit but devoid of the typical 2',5'-OAS catalytic activity have been described. These OAS-related proteins are encoded by a gene located at the proximity of the 2',5'-OAS locus. These findings illustrate the apparent structural and functional complexity of the human 2',5'-OAS family.

The expression of both domains of the 69/71 kDa 2',5' oligoadenylate synthetase generates a catalytically active enzyme and mediates an anti-viral response.

The 2',5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which, when activated by double-stranded (ds) RNA, polymerizes ATP into 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, where n >/= 1. The 69-kDa form of human OAS has two isoforms (p69 and p71) that are identical for their first 683 amino acids and consist of two homologous and adjacent domains, each homologous to the small 40-kDa OAS. Here, we demonstrate that mRNA species specific for the isoforms p69 and p71 are enhanced in interferon-treated cells, with the p69 mRNA being more abundant than that of p71. In transfected cells, both isoforms could be expressed independently to generate enzymes with similar catalytic activity, typical of the natural 69-kDa OAS from interferon-treated cells. On the other hand, deletion mutants expressing either the N- or C-terminal domain common in p69 and p71 were greatly unstable and were found to be devoid of catalytic activity, in spite of the capacity of the C-terminal domain to bind dsRNA. Finally, we show that murine cell lines stably expressing either p69 or p71 isoforms partially resist infection by the encephalomyocarditis virus. These results indicate that both isoforms of the 69-kDa form of 2',5' OAS are expressed in interferon-treated cells, and that each isoform could be implicated in the mechanism of the anti-viral action of interferon.

The V3 loop-mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP inhibits HIV infection in primary macrophage cultures.

The V3 loop-mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP [psi(CH2N) representing a reduced peptide bond], which presents pentavalently the tripeptide Kpsi(CH2N)PR, is a potent inhibitor of HIV entry. By its capacity to bind specifically protein components on the cell surface, 5[Kpsi(CH2N)PR]-TASP blocks the attachment of virus particles to permissive CD4+ cells. Here, the inhibitory effect of 5[Kpsi(CH2N)PR]-TASP was investigated in monocyte-derived macrophages (MDMs) infected by the monocytotropic HIV-1(Ba-L) isolate. We show that 5[Kpsi(CH2N)PR]-TASP inhibits HIV-1(Ba-L) infection in a dose-dependent manner, with more than 90% inhibition at 2 microM concentration. On the other hand, the control 5[QPQ]-TASP construct and the monovalent Kpsi(CH2N)PR tripeptide have no effect even at high concentrations. Under such experimental conditions, the biotin-labeled 5[Kpsi(CH2N)PR]-TASP, but not the Kpsi(CH2N)PR construct, binds specifically to the surface of MDMs and forms a stable complex with the cell surface-expressed nucleolin, as has been demonstrated to be the case in peripheral blood mononuclear cells. Infection of MDMs by HIV-1(Ba-L) could also be inhibited by beta-chemokines RANTES and MIP-1beta. Interestingly, association of low concentrations of 5[Kpsi(CH2N)PR]-TASP and beta-chemokines results in a synergistic inhibitory effect on HIV infection compared with the effect observed with each reagent alone. The inhibitory effect of 5[Kpsi(CH2N)PR]-TASP in primary macrophage cultures point out its potential as an anti-HIV drug in cells, which are the natural viral targets.

The 100-kDa 2',5'-oligoadenylate synthetase catalyzing preferentially the synthesis of dimeric pppA2'p5'A molecules is composed of three homologous domains.

The 2-5A synthetases represent a family of proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, n >/= 1. Three forms of human 2-5A synthetases have been described corresponding to proteins of 40/46 (p40/p46), 69/71 (p69/p71), and 100 kDa (p100). Here we describe the molecular cloning and characterization of p100. By screening a cDNA expression library with a specific p100 polyclonal antibody, we first isolated a 590-nucleotide cDNA fragment which was subsequently used to isolate the full-length 6365-nucleotide cDNA. This cDNA recognizes a distinct interferon-induced messenger RNA of 7 kilobases. It has an open reading frame encoding a protein of 1087 amino acids including the sequence of seven peptides obtained by microsequencing of the natural p100 protein, which was purified from interferon-treated human cells. p100 is composed of three adjacent domains, each homologous to the previously defined catalytic unit of 350 amino acids, which is present as one unit in p40/p46 and as two units in p69/p71. The recombinant p100 synthesized preferentially dimeric 2', 5'-oligoadenylate molecules and displayed parameters for maximum enzyme activity similar to the natural p100. These results confirm that the enzymatic activity of p100 is distinct compared with that of p40/p46 and p69/p71.

Molecular cloning and characterization of two related and interferon-induced 56-kDa and 30-kDa proteins highly similar to 2'-5' oligoadenylate synthetase.

The 2'-5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which when activated by double-stranded (ds) RNA polymerizes ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n> or =1. Three forms of human OASs have been described corresponding to proteins of 40/46, 69/71 and 100 kDa (p40/p46, p69/p71 and p100). Polyclonal antibodies raised against p100 cross reacted with the other forms p40/p46, p69/p71 but also with an interferon-induced 56-kDa protein (p56). By screening a cDNA expression library, these polyclonal antibodies selected a cDNA encoding p56. Further studies by the RACE procedure using primers corresponding to this cDNA, a p56-related protein of 30 kDa (p30) was isolated. Both p56 and p30 mRNA are expressed in interferon-treated cells as transcripts of 2 kb and 1.8 kb, respectively. The 1.8-kb mRNA is homologous to the 2-kb mRNA but with a 243-nucleotide deletion at position 1011, which results in a frameshift. Consequently, the p56 and p30 have their first 219 amino acid residues identical but differ at their C-termini. In vitro transcription-translation of p56 and p30 cDNAs generated proteins of 56 and 30 kDa, respectively. The deduced amino acid sequence of p56 kDa shares strong similarity with the previously cloned OASs, and contains the subdomains conserved in p40/p46 and p69/p71 forms. Transient expression in HeLa cells indicated that p30 has a cytoplasmic localization, whereas p56 has cytoplasmic and nucleolar localizations. The p56 isolated from transfected cells was shown to bind dsRNA and DNA, but it was devoid of 2'-5'OAS activity typical of the three known forms of this enzyme. Thus, p56 and p30 are two related and interferon-induced proteins outside the family of 2'-5'OAS, which might have as yet unidentified catalytic activities or functions.

The human 2',5'-oligoadenylate synthetase locus is composed of three distinct genes clustered on chromosome 12q24.2 encoding the 100-, 69-, and 40-kDa forms.

The 2',5'-oligoadenylate synthetases (OAS) represent a family of interferon-induced proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, n >/= 1. Three forms of human OAS corresponding to proteins of 40/46, 69/71, and 100 kDa have been described. Based on the deduced amino acid sequences of the corresponding cDNAs, these OAS share a homologous region of about 350 amino acid residues that could represent the functional domain of OAS; the 40/46 proteins contain one single domain, whereas the 69/71- and the 100-kDa proteins contain two and three adjacent domains, respectively. Here we show that the cDNAs for OAS-40/46, OAS-69/71, and OAS-100 hybridize to distinct interferon-induced mRNAs of 2 kb; 2.8, 3.3, 3.9, and 4.5 kb; and 7 kb, respectively. By in situ hybridization, we assign the human OAS-40/46, OAS-69/71, and OAS-100 genes (referred to as OAS1, OAS2, and OAS3, respectively) to a unique cytogenetic location on chromosomal region 12q24.2. We constructed a YAC, PAC, and cosmid contig carrying the three OAS genes and provide evidence that the three genes are clustered within a single PAC clone of 130 kb. The three OAS genes are flanked by markers WI-10614 (cen) and D12S2293 (tel) and are contained within three sets of overlapping cosmid clones. They share the same orientation of transcription and are arranged in the order cen- 5'-OAS1-OAS3-OAS2-3'-tel. We suggest that clustering of these genes reflects their evolutionary relationship possibly through the duplication of the conserved functional domain. This ready-to-sequence PAC and cosmid contig provides a valuable tool for identifying regulatory elements involved in the transcription of the OAS genes when induced by interferon and for elucidating the exon-intron organization of these genes.

Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26.

Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.

Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells.

The binding of human immunodeficiency virus (HIV) type 1 particles to CD4(+) cells could be blocked either by antibodies against the V3 loop domain of the viral external envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kpsi(CH2N)PR]-TASP and cytoplasmic extracts from human CEM cells, we purified three V3 loop-binding proteins of 95, 40, and 30 kDa, which after microsequencing were revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is inhibited either by 5[Kpsi(CH2N)PR]-TASP or antibodies against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4(+) cells as efficiently as soluble CD4. Taken together, our results suggest that nucleolin, PHAP II, and PHAP I appear to be functional as potential receptors in the HIV binding process by virtue of their capacity to interact with the V3 loop of gp120.

Increased rate of HIV-1 entry and its cytopathic effect in CD4+/CXCR4+ T cells expressing relatively high levels of CD26.

The role of the T-cell activation antigen CD26 was evaluated in viral entry and infection of CD4(+)/CXCR4(+) cells by the lymphotropic HIV-1 Lai isolate. For this purpose, CEM T cells, which are permissive to HIV infection and express low levels of CD26, were used to establish by transfection four groups of cell clones expressing either low, high, and very high levels of CD26, or expressing the anti-sense RNA of CD26. Entry was monitored by the detection of proviral DNA synthesis and the kinetics of virus production, whereas the cytopathic effect was demonstrated by the occurrence of apoptosis. HIV entry and infection were consistently accelerated by at least 24 to 48 h in clones expressing high levels of CD26 compared to the parental cells or to the clones expressing low levels of CD26. Interestingly, infection of clones expressing very high levels of CD26 was not accelerated and showed a kinetics of infection similar to that of low CD26 expressing clones. Moreover, HIV infection was significantly reduced in the clones expressing CD26 anti-sense RNA. In the different clones, apoptosis was dependent on the severity of virus infection and occurred after the accumulation of HIV envelope glycoproteins. Our results demonstrate that with equivalently expressed levels of CD4 and CXCR4 in cell lines established from CEM cells, relatively high levels of CD26 contribute to an increased rate of HIV entry, infection, and apoptosis. Furthermore, they point out that overexpression of CD26 in a given cell line may lead to a negative effect on HIV infection. Consequently, CD26 appears to regulate HIV entry and apoptosis, processes which are critical for viral pathogenesis.

Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26.

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.