Measurement of plasma HIV-1 RNA below the limit of quantification (<20 copies/mL) of commercial assays with the integrase HIV RNA single-copy assay.

BACKGROUND: Plasma HIV-1 RNA (viral load, VL) is measured routinely in HIV-infected persons with FDA-approved commercially available assays such as the Cobas-TaqMan HIV-1 Assay v2.0. This assay provides quantification of viremia ≥20 copies/mL. More sensitive methods, able to quantify low-level persistent viremia below the detection limit of commercially available assays, are needed to assess the impact of current HIV cure strategies on viremia. OBJECTIVES: The novel integrase HIV-1 RNA single-copy assay (iSCA) was evaluated for measurement of low-level persistent viremia in clinical trial samples (n = 151) from subjects participating in Gilead HIV clinical research. STUDY DESIGN: Paired plasma samples from HIV-1-infected patients treated with combination ART were assessed using both HIV-1 Cobas-TaqMan and iSCA; results from the two assays were compared. RESULTS: Paired Cobas-TaqMan/iSCA data were obtained for 151 HIV-infected adults. Most samples (117/151, 77%) had non-quantifiable Cobas-TaqMan result, either <20 copies/mL ("<20") or "Target Not Detected" (TND). All 117 non-quantified samples were quantified with iSCA and showed higher HIV-1 RNA levels in samples with <20 than TND Cobas-TaqMan results (p < 0.0001). CONCLUSIONS: In this large sample collection from virologically suppressed HIV-infected adults, use of iSCA led to quantification of low-level viremia below the limit of detection of the Cobas-TaqMan assay in all 117 previously non-quantifiable plasma samples. These data confirm the value of the iSCA as a helpful addition to the classical HIV VL assays and its potential for use in HIV cure studies to assess whether experimental interventions alter viremia.

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.

CD8(+)-Cell antiviral factor activity is not restricted to human immunodeficiency virus (HIV)-specific T cells and can block HIV replication after initiation of reverse transcription.

CD8(+) lymphocytes from human immunodeficiency virus (HIV)-infected patients can suppress in vitro HIV replication in CD4(+) T cells by a noncytolytic mechanism involving secreted CD8(+)-cell antiviral factor(s) (CAF). Using an HIV Nef-specific cytotoxic-T-lymphocyte (CTL) line and autologous CD4(+) T cells infected with a nef-deleted HIV-1 virus, we demonstrated that, after a priming antigenic stimulation, this suppression does not require the presence of the specific antigen during the effector phase. Furthermore, using an Epstein-Barr virus (EBV)-specific CTL line from an HIV-seronegative donor, we demonstrated that the ability to inhibit HIV replication in a noncytolytic manner is not restricted to HIV-specific effector cells; indeed, EBV-specific CTL were as efficient as HIV-specific effectors in suppressing R5 or X4 HIV-1 strain replication in vitro. This HIV-suppressive activity mediated by a soluble factor(s) present in the culture supernatant was detectable for up to 14 days following stimulation of EBV-specific CD8(+) cells with the cognate epitope peptide. Following acute infection of CEM cells with an X4 strain of HIV-1, EBV-specific CTL line supernatant containing HIV-suppressive activity did not block virus entry but was shown to interfere with virus replication after the first template switching of reverse transcription. Our results suggest that the noncytolytic control of HIV replication by EBV-specific CD8(+) T lymphocytes corresponded to a CAF-like activity and thus demonstrate that CAF production may not be restricted to CTL induced during HIV disease. Moreover, CAF acts after reverse transcription at least for X4 isolate replication inhibition.

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.

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.

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.

Adenosine deaminase binding to human CD26 is inhibited by HIV-1 envelope glycoprotein gp120 and viral particles.

CD26, known to be the adenosine deaminase (ADA)-binding protein, has been implicated in HIV infection. Several studies have revealed a correlation between depletion of CD4+/CD26+ T lymphocytes, increased serum levels of ADA, and the evolution of AIDS in infected individuals. We show that in human B and T cell lines, irrespective of CD4 expression, 125I-labeled ADA binding to CD26 is inhibited by recombinant soluble HIV-1 envelope glycoprotein gp120 and by HIV-1 infectious particles. Accordingly, an anti-CD4 mAb, which inhibits the binding of gp120 to CD4 and blocks viral infection, did not affect inhibition of 125I-labeled ADA binding to CD26 by HIV particles. On the other hand, mAbs directed against the V3 loop and the C-terminal region of gp120 abolished completely the inhibitory effect. Overlapping synthetic peptides covering the entire gp120 sequence were tested to map the region in gp120 responsible for ADA binding inhibition. Only peptides in the C3 region significantly inhibited the binding of ADA to CD26. These results provide indirect evidence for the interaction of gp120 with CD26 and indicate that a specific function of gp120 is the inhibition of ADA binding to CD26 in both CD4+ and CD4- cells. Because ADA deficiency leads to severe combined immunodefiency syndrome, it remains possible that HIV particle-mediated blockade of ADA-CD26 interaction may have significant consequences in the pathogenesis of AIDS.

Pseudopeptide TASP inhibitors of HIV entry bind specifically to a 95-kDa cell surface protein.

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Further characterization of DPP IV-beta, a novel cell surface expressed protein with dipeptidyl peptidase activity.

By using a CD26 negative human lymphoblastoid cell line (C8166), here we describe the characterization of a cell-surface protein which manifests CD26-like dipeptidyl peptidase IV (DPP IV) activity. This protein, referred to as DPP IV-beta, shows a higher KM value for Gly-Pro-pNA than CD26 (0.31 mM compared to 0.11 mM, respectively). In addition, DPP IV-beta was found not to bind 125I-labeled adenosine deaminase (a property of human CD26). Gel filtration experiments using extracts from C8166 and MOLT4 (a CD26 positive human T cell line) cells, revealed that the apparent molecular mass of DPP IV-beta is 82 kDa, whereas that of CD26 is 110 kDa. In order to conveniently differentiate both activities, a new family of inhibitors, that selectively blocks peptidase activity associated to CD26, has been developed.

CD26 as a positive regulator of HIV envelope-glycoprotein induced apoptosis in CD4+ T cells.

The membrane-expressed HIV-1 envelope glycoprotein complex, gp120/gp41, has been shown to be responsible for the initiation of cell killing by apoptosis in CD4+ T cells. By using two experimental approaches we demonstrate that CD26, independent of its DPP IV activity, appears to be implicated in this function of the gp120/gp41 complex to initiate apoptosis.

The level of CD26 determines the rate of HIV entry in a CD4+ T-cell line.

We have reported that CD26 could serve as a cofactor of CD4 in HIV entry. Recently, more evidence has been provided for the implication of CD26 in HIV entry, replication and cytopathic effect. Along with, we have demonstrated that the level of CD26 may determine the rate of HIV-envelope induced-apoptosis. The role of CD26 in HIV entry was further investigated using CEM T-cell line. Clones were established by transfection, expressing different levels of CD26. Entry, infection and cytopathic effect were monitored in several independent clones, and were found to be delayed in clones CD26-Low and CD26-SuperHigh compared to clones CD26-High. The delay was most significant in clones CD26-AntiSense, without any apparent cytopathic effect. These results demonstrate that relatively enhanced levels of CD26 contribute to an increased virus infection. Furthermore, they illustrate that CD26-SuperHigh clones manifest a phenotype similar to CD26-Low clones. This point out the critical role of CD26 in the rate of HIV entry and its cytopathic effect, two events which are initiated by the interaction of HIV envelope glycoproteins with cell-surface CD4.

HIV-1 envelope gp120 and viral particles block adenosine deaminase binding to human CD26.

CD26, known to be the adenosine deaminase (ADA) binding protein, has been implicated in HIV infection. In human B and T cell lines we show that, irrespective of CD4 expression, 125I-labeled ADA binding to CD26 is inhibited by recombinant soluble HIV-1 envelope glycoprotein gp120 and by HIV-1 infectious particles. Overlapping synthetic peptides covering the entire gp120 sequence were tested to map the region in gp120 responsible for ADA binding inhibition. Only peptides in the C3 region significantly inhibited the binding of ADA to CD26. These results indicate that a specific function of gp120 is the inhibition of ADA binding to CD26 in both CD4+ and CD4- cells. Since the interaction ecto-ADA/CD26 is required for the activation of T cells, it remains possible that HIV particle-mediated blockade of ecto-ADA/CD26 interaction may have significant consequences in the pathogenesis of AIDS disease.

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.

HIV envelope glycoprotein-induced cell killing by apoptosis is enhanced with increased expression of CD26 in CD4+ T cells.

The membrane-expressed HIV-1 envelope glycoprotein complex, gp120 and gp41, has been shown to be responsible for the initiation of cell killing by apoptosis in CD4+ T cells. By using two experimental approaches we demonstrate here that CD26, also known as dipeptidyl peptidase IV (DPP IV), appears to be implicated in this function of the gp120/gp41 complex to initiate apoptosis. In the first experimental model, we used persistently HIV-1-infected H9/IIIB cells expressing the membrane-associated gp120/gp41 complex as effector cells to induce apoptosis in Jurkat CD4+ T cells: parental or transfected in order to express high levels of recombinant CD26, either wild-type or mutated at its Ser-630 which inactivates the DPP IV activity of CD26. Parental Jurkat cells and transfected control cell clones express low but reproducibly detectable levels of endogenous CD26. In coculturing experiments using H9/IIIB and Jurkat cells, the occurrence of apoptosis was found to be retarded by at least 24 hr in Jurkat cells expressing low levels of endogenous CD26, compared to cell clones expressing high levels of either wild-type or mutated catalytically inactive transfected CD26. In the second experimental model, the different Jurkat cell lines were infected with vaccinia recombinant viruses expressing HIV-1 env gene, either wild-type to generate a functional gp120/gp41 complex or mutated to generate an uncleavable membrane-expressed precursor of the envelope glycoprotein gp160. At 18 hr postinfection with such vaccinia recombinant viruses, apoptosis was observed only in Jurkat cells with enhanced levels of CD26 and expressing the gp120/gp41 complex. Apoptosis was not detected in the different Jurkat cell lines expressing the uncleavable precursor gp160. In both of the experimental models used, no significant differences were observed between the transfected cells expressing either the wild-type or the mutated form of CD26, thus suggesting that the DPP IV activity of CD26 is not essential for its function as a cofactor of CD4 in the mechanism of initiation of apoptosis by the HIV envelope gp120/gp41 complex. Taken together, these results indicate that CD26 in CD4+ T cells may determine the rate of initiation of apoptosis by the mature HIV-1 envelope glycoproteins, i.e., CD26 is being involved as a cofactor of CD4 in the mechanism of triggering apoptosis by the gp120/gp41 complex. As signaling through CD26 could lead to T cell activation, we propose that this latter might be modified following the binding of the gp120/gp41 complex to CD4 and thus leading to apoptosis.

Dipeptidyl-peptidase IV-beta, a novel form of cell-surface-expressed protein with dipeptidyl-peptidase IV activity.

The T-cell activation antigen CD26, is a type II membrane glycoprotein with intrinsic dipeptidyl-peptidase IV (DPP IV) activity, characterized by its capacity to cleave off N-terminal dipeptides containing proline as the penultimate residue. Independent of its catalytic activity, CD26 has also been characterized as adenosine deaminase binding protein. By using CD26 negative human C8166 cells, here we describe the existence of another cell-surface protein which manifests CD26-like DPP IV activity. For convenience, this protein will be referred to as DPP IV-beta. Consistent with the cell-surface expression of DPP IV-beta, intact C8166 cells manifested a high level of DPP IV, whereas, they manifested poor activity against substrates of DPP II known to have an intracellular localization. A partially purified preparation of CD26 from human MOLT4 cells, and the DPP IV-beta expressed on intact cells were found to possess similar catalytic activity and pH optimum. In addition, cell-surface CD26 and DPP IV-beta on intact MOLT4 and C8166 cells, respectively, resisted digestion by proteolytic enzymes such as trypsin and proteinase K. However, adenosine deaminase activity was not detectable on the surface of C8166 cells in contrast to CD26 positive MOLT4 cells. In accord with this, 125I-labeled adenosine deaminase which binds CD26 was found not to bind DPP IV-beta. Gel-filtration experiments using 0.5% Triton X-100 extracts from C8166 and MOLT4 cells, revealed that the apparent molecular mass of DPP IV-beta is 82 kDa, whereas that of CD26 is 110 kDa as expected. Taken together, our results suggest that DPP IV-beta is a CD26-like protein which could be characterized by distinct properties.