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International audience; CBD1 peptide (SLEQIWNNMTWMQWDK), corresponding to the consensus caveolin-1 binding domain in HIV-1 envelope glycoprotein gp41, elicits the production of antibodies that inhibit infection of primary CD4(+) T lymphocytes by various primary HIV-1 isolates. Here the immunogenicity of the CBD1 peptide was investigated in cynomolgus macaques using adjuvants that are acceptable for human use. In the first set of studies, macaques were immunized with the CBD1 peptide in association with muramyl dipeptide derivative MDP-Lys(L18) combined with the oil-in-water emulsion, MF-59. After five immunizations at 4 weeks interval, the antibody titer against the CBD1 peptide was found to be either medium, poor, weak or none, thus suggesting that the CBD1 immune response might be restricted by the major histocompatibility complex (MHC) class II molecules. In the second set of studies therefore, macaques were immunized with the CBD1 peptide in association with the 'promiscuous' T cell epitope from the tetanus toxin, either as free peptides or covalently linked with the dilysine linker using CpG ODN and Montanide ISA 51 as adjuvants. This latter immunization procedure boosted markedly the anti-CBD1 antibody response, since even the non-responders generated high-titered peptide-specific antibodies. Moreover, co-immunization of the CBD1 and the T helper epitope as free peptides seemed to be favorable for the production of neutralizing antibodies, with 50% inhibition of HIV-1 infection occurring at 300-400-fold dilution of the immune sera. Finally, neutralizing and non-neutralizing immune macaque sera could be differentiated by the profile of cross-reactivity with overlapping CBD1-related peptides in ELISA. Taken together, our results demonstrate that the CBD1 peptide is immunogenic in macaques and that an eventual MHC-restriction could be overcome by the administration with an appropriate T helper epitope. more...

Caveolin-1 is a scaffolding protein that organizes and concentrates specific ligands within the caveolae membranes. We identified a conserved caveolin-1 binding motif in the HIV-1 transmembrane envelope glycoprotein gp41 and designed several synthetic peptides, referred to as CBD1, corresponding to the consensus caveolin-1 binding domain in gp41. In rabbits, these peptides elicit the production of antibodies that inhibit infection of primary CD4+ T lymphocytes by various primary HIV-1 isolates. Interestingly, gp41 exists as a stable complex with caveolin-1 in HIV-infected cells. Anti-CBD1 peptide antibodies, therefore, might be functional by inhibiting the potential interaction of gp41 with caveolin-1. Because of their capacity to elicit antibodies that inhibit the different clades of HIV-1, CBD1-based peptides may represent a novel synthetic universal B cell epitope vaccine candidate for HIV/AIDS. Moreover, such peptides could also have an application as a therapeutic vaccine since CBD1-specific antibodies are rare in HIV-infected individuals from several geographic origins. more...

The cross-linking of HIV on permissive cells results aggregation of HIV particles with surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin coaggregate with glycolipid-enriched membrane microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEM-associated proteins. After HIV entry, nucleolin is recovered also in fractions containing HIV DNA, viral matrix, and reverse transcriptase, thus suggesting that it could accompany viral entry. We show that surface nucleolin is markedly down-regulated a few hours following HIV entry into permissive cells; an effect that appears to be the consequence of its translocation into the cytoplasm. Our findings demonstrate that anchorage of HIV particles on permissive cells induces aggegation of surface nucleolin and its association with detergent-insoluble lipid raft components. Moreover, they support the suggestion that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process. more...

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. more...

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. more...

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-1envgene, 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. more...

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients. more...

The mechanism of action of different types of interferons (IFN-alpha, -beta, and -gamma) against human immunodeficiency virus (HIV)-1 infection was investigated in chronically infected monocytoid U937 cells and during an acute infection of the T lymphoblastoid CEM cells. Two chronically infected U937 cell populations, obtained independently (referred to as type A and B cells), were analyzed for their response to IFNs. In type A cells, IFNs mainly inhibited virus particle release, whereas in type B cells, the anti-HIV effect of IFNs cells was found to be largely due to a specific inhibition of viral protein synthesis without any apparent effect on total cellular protein synthesis. Interestingly, such a differential inhibition of HIV protein synthesis could also be demonstrated in acutely infected CEM cells in response to treatment with IFN-alpha. Both in chronically infected U937 type B and acutely infected CEM cells, equivalent amounts of nuclear and cytoplasmic HIV-1 mRNA were detected in control and IFN-treated cells in spite of at least 80% inhibition of HIV protein synthesis. Analysis of the distribution of cellular and viral mRNAs on polysomes in HIV-1-infected cells demonstrated that IFN treatment induces a specific block on viral mRNA translation. These results indicate that the antiviral mechanism of IFN on later stages of HIV replication cycle may be partly due to the inhibition of HIV mRNA translation, besides an effect on virus budding or release. more...

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process. more...

Interferon-treated mouse cells show an enhanced level of protein kinase activity which is manifested by the phosphorylation of an endogenous 67,000-molecular weight protein (p67K kinase). This kinase activity can be assayed efficiently after its partial purification on poly(I) · poly(C)-Sepharose. We have previously shown that the p67K kinase is present in the liver, spleen and plasma (heparinized) of mice with high levels of circulating interferon. Here we confirm these results by treatment of mice with interferon and furthermore show that besides the liver and spleen, the level of p67K kinase is enhanced in several other tissues such as thymus, brain, pancreas, heart, and lung. The action of interferon in mice was further monitored by the assay of pppA(2′p5′A)n synthetase (2–5A synthetase) in different tissues. The level of 2-5A synthetase was enhanced several fold in the following tissues: heart, pancreas, thymus, liver, and spleen. The detection of 2–5A synthetase and p67K kinase activities in the different tissues of mice provides suitable markers for the response of each individual tissue toward treatment with interferon. The phosphorylated 67K protein (pp67K) from control and interferon-treated mouse L-929 cells and from the plasma and different tissues of control and interferontreated mice was characterized by two-dimensional gel electrophoresis. The isoelectric point ( pl ) of pp67K from the different tissues and L-929 cells was 8 to 8.5. On the other hand the p I of pp67K from the plasma had a range of 7.5 to 8. These results indicated that the presence of p67K kinase in the plasma of mice is not due to lysis of tissue cells. more...

Treatment of patients with interferon or inducers of interferon results in an enhanced level of a protein kinase activity found in platelets (1,3). The kinase activity is responsible for the phosphorylation of a 70–72,000 molecular weight protein (72K protein) found in blood plasma. By the means of a technique based on the precipitation of this protein kinase system (the protein kinase and its substrate), we show here that the 72K protein is the α-chain of fibrinogen. During the coagulation process induced by thrombin, the 32P-labelled 72K protein is recovered in the clot. After incubation in the presence of thrombin, the 72K protein looses a small polypeptide of 2–3000 in molecular weight resulting a shift in its isoelectric point (pI) from 6.8–7.0 to 7.5. At the end of the coagulation process, the 32P-labelled 72K protein becomes undetectable since it gives rise to a covalently linked α-polymer of a high molecular weight. In accord with these results, the 72K protein could be precipitated by antibodies against human fibrinogen. more...

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous 67,000 and 72,000 Mr proteins, respectively. Enhanced levels of such kinase activity are also detectable in the plasma of patients treated with interferon and in the plasma and tissues of interferon-treated mice. A rapid and efficient method of assay for these protein kinase activities is described. The samples are first incubated with heparin (100 units/ml), which results in the inhibition of different protein kinase activities, but not the one mediated by interferon. The latter one is then assayed after partial purification on poly(rI):(rC)-Sepharose or poly(rG)-Sepharose. The protein kinase from human and mouse cells in culture and from the different tissues of mice binds specifically to poly(rI):(rC)-Sepharose. On the other hand, the protein kinase activity from both mouse and human plasma shows a higher affinity toward poly(rG)-Sepharose. These methods are successfully applied for the determination of the interferon-mediated protein kinase activity from tissue extracts and plasma. more...

A new isolate of the human immunodeficiency virus (HIV) related to the HIV-2 strain was isolated from peripheral blood lymphocytes of an Ivory Coast patient with AIDS. This isolate referred to as HIV-2 EHO could be differentiated by its envelope precursor and external glycoprotein which are 20-kDa smaller than those of HIV-2 ROD isolate. Furthermore, the apparent molecular weight of the major core protein of HIV-2 EHO is 27 kDa instead of 26 kDa as in HIV-2 ROD isolate. In addition, the product of the vpx gene which is a characteristic feature of the HIV-2 strain, is 14 kDa in HIV-2 EHO compared with 16 kDa in HIV-2 ROD. In contrast to these, the envelope precursor of HIV-2 EHO forms a transient dimer during its maturation as is the case for HIV-2 ROD. In both cases the transmembrane proteins are 36 kDa and exist as homodimers of 80 kDa. Endoglycosidase H digestion experiments indicated that the 20-kDa difference between the two HIV-2 isolates is not due to a difference in the number of N-linked oligosaccharide chains per polypeptide, since deglycosylated envelope precursors of HIV-2 ROD and EHO have an apparent molecular weight of 80 and 60 kDa, respectively. Partial proteolysis of the envelope precursors from the two isolates with Staphylococcus aureus V8 protease gave a distinct pattern of polypeptides. These results suggest that the differences between the external envelope proteins of the two HIV-2 isolates are due to their amino acid composition. Accordingly, polyclonal antibodies raised against HIV-2 ROD envelope do not recognize the corresponding envelope proteins of HIV-2 EHO by immunoblotting and immunoprecipitation assays. These data illustrate that analysis of viral proteins could be useful for a rapid characterization of new viral isolates and show the heterogeneity of HIV-2 isolates in West Africa. more...

Summary Interferon-treated human cells show an enhanced level of a double-stranded (ds) RNA-dependent protein kinase activity which is manifested by the phosphorylation of an endogenous 72,000 molecular weight protein (p72K kinase). By the use of murine polyclonal antibodies against this p72K kinase, here we have characterized the protein kinase activity associated with immune complexes precipitated from extracts of interferon-treated cells. Precipitation of the p72K kinase by the polyclonal antibodies results in the formation of a complex in which the kinase activity is manifested by phosphorylation of the 72K protein. This phosphorylation, however, is independent of dsRNA. Such immunoprecipitates can also phosphorylate exogenous substrates, calf thymus histones and the α subunit of protein initiation factor eIF2. more...

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous Mr = 67,000 and 72,000 proteins, respectively. Such kinase activity can be assayed after its partial purification on poly(I) X poly(C)-Sepharose. Under these experimental conditions, the apparent km of the kinase for ATP is 1.0 X 10(-6) M and 2.5 X 10(-6) M in enzyme fractions from mouse L-929 and human HeLa cells, respectively. The Mr = 67,000 and 72,000 proteins are phosphorylated by their serine and threonine residues, the ratio of which is modified in preparations from interferon-treated cells. Both of these phosphoproteins are composed of several subspecies with similar isoelectric points (pIs) in the range of 7.2 to 8.2. This heterogeneity is due to the number of phosphate groups per molecule of protein. Accordingly, the pIs of highly phosphorylated proteins are at a less basic pH (7.2 to 7.5). Furthermore, highly phosphorylated proteins show an increase in their apparent molecular weights compared to partially phosphorylated ones. This corresponds to an increase of Mr = 1,500. Partial proteolysis of the 32P-labeled Mr = 67,000 and 72,000 proteins by Staphylococcus aureus V8 protease, alpha-chymotrypsin and thrombin, indicated that these phosphoproteins differ in their polypeptide structure. Phosphorylation of the Mr = 67,000 and 72,000 proteins in enzyme fractions from control L-929 and HeLa cells is enhanced by mixing with extracts from interferon-treated heterologous cells. Proteins, Mr = 67,000 and 72,000, therefore, may serve as suitable substrates for an exogenous kinase, thus indicating that the substrate in enzyme fractions from control cells is less phosphorylated because of a low level of kinase activity. more...