Human Immunodeficiency Virus Gp120 Research Articles

The M22 antibody identifies highly activated reactive astrocytes responding to central nervous system disease.

Astrocytes respond vigorously to diverse neurological insults. It is still not clear, however, whether this response is stereotypic following different insults or varies according to the injury. We have used a novel immunocytochemical marker of reactive astrocytes, termed M22, together with antibodies to glial fibrillary acidic protein (GFAP), to analyze region- and insult-specific differences in reactive astrocytosis in the murine central nervous system (CNS). Pathology was variously induced by (1) infectious agents, (2) transgenic overexpression of a viral glycoprotein or cytokine, or (3) focal trauma. Scrapie infection induced high levels of both GFAP and M22 epitope expression by hippocampal reactive astrocytes, but neither scrapie nor wild mouse retrovirus infection induced detectable M22 staining in reactive astrocytes of the caudal brain. Focal trauma and human immunodeficiency virus gp120 overexpression induced M22 expression only in the hippocampus, while interleukin-6 overexpression induced it in cerebellar astrocytes. Although M22 expression was limited to areas with extensive damage, GFAP expression was induced in every region of the mouse brain displaying pathology. Staining of routinely fixed human brain tissue demonstrated that M22 also labeled reactive astrocytes in chronic human CNS disease. The restriction of M22 expression to areas of strongly GFAP-positive astrocytosis suggests that the M22 antibody identified highly activated reactive astrocytes. Because of this selective staining of activated astrocytes, the M22 antibody may provide neuropathologists with a good marker for qualitative analysis of the astrocytic response to different injuries.

Antibody-producing effects in mice by synthetic immunoactive lipopeptides with the conjugated amino acid sequence of gp120 in human immunodeficiency virus.

To induce peptide-specific antibodies in mice, as a model for vaccination against human immunodeficiency virus (HIV), lipopeptide analogs conjugated with three repeating units (KAB-112; designated as gp120-peptide) of a part (Gly-Pro-Gly-Arg-Ala-Phe) of the amino acid sequences of the V3 loop region in gp120 of HIV were synthesized. The mitogenicity, production of nitric oxide (NO) and induction of peptide-specific antibodies in mice by synthetic lipopeptides were examined. Compounds, KAB-8 (diacylglycerol-tetrapeptide having a part of the amino acid sequence in Escherichia coli), KAB-116 (diacylglycerol-cysteine), KAB-117 (diacylglycerol with gp120-peptide) and KAB-121 (KAB-8 with gp120-peptide) were capable of increasing significantly the incorporation of [3H]thymidine into splenocytes of C3H/He mice at concentrations ranging from 12.5 to 100 microM, but KAB-112 (gp120-peptide) and KAB-115 (monoacylglycerol with gp120-peptide) did not show such activity. The compounds, KAB-8, KAB-117 and 121, exhibited NO production in murine macrophages. When 50 nmol of these compounds was administered intraperitoneally into BALB/c mice on days 0, 16 and 46, the peptide-specific antibody titers in their sera produced by each compound were determined with ELISA. The sera of KAB-117 and KAB-121, which were obtained on days 14, 30, 42, 57 and 70, had a higher titer than that of KAB-112 and KAB-115, suggesting that the diacylglycerol derivative enhance the production of the peptide-specific antibodies.

Maturation and assembly of retroviral glycoproteins.

Retroviruses infect cells after binding to a diverse set of specific cell surface receptors via their envelope glycoproteins (Env) (reviewed in (Weiss 1993). Retroviralenv genes are translated into a precursor protein that enters the secretory pathway of the host cell. During the process of being transported to the plasma membrane, the site at which the glycoproteins become incorporated into virions, the precursor undergoes maturation to a bipartite complex composed of the N-terminal, external surface subunit (SU), and a membrane-spanning Cterminal protein (TM). The receptor specificity of the complex is determined in large part, if not exclusively, by the SU domain, which retains receptor binding activity outside of the complex with T M (Delarco and Todaro 1976; Lasky et al. 1987). The diversity of receptor specificities within some groups of retroviruses has facilitated the identification of regions of the SU proteins that contribute to receptor binding. The heterogeneity of SU proteins that is apparent fromenv sequences of different retroviruses suggests that the structural details of Env-receptor interactions may vary among these viruses. In addition, the sequences or domains that have been implicated in receptor binding differ in their positions within SU. The gp70 of the murine leukemia viruses (MuLVs) appears to be organized into N-terminal and C-terminal structural domains with the receptor-binding determinants in the N-terminal region (Battini et al. 1992; Heard and Danos 1991; Morgan et al. 1993; Ott and Rein 1992), while receptor specificity of avian sarcoma and leukemia virus (ASLV) Env is dependent on sequence in the middle third of S U (Bova et al. 1986, 1988; Dorner and Coffin 1986; Dorner et al. 1985). Receptor binding by gp120 of human immunodeficiency virus (HIV-1) involves a major determinant in the C-terminal third of the protein together with discrete segments from other regions of the protein (Cordonnier et al. 1989; Dowbenko et al. 1988; Lasky et al. 1987; Olshevsky et al. 1990; Pollard et al. 1992). Whether these differences extend to the structures of the SU proteins or only indicate variety in the interaction of a relatively conserved structure with different receptors is unclear.

N-Acetylcysteine Enhances Antibody-Dependent Cellular Cytotoxicity in Neutrophils and Mononuclear Cells from Healthy Adults and Human Immunodeficiency Virus-Infected Patients

Patients with AIDS have decreased levels of the intracellular antioxidant, glutathione, in their circulating lymphocytes and plasma. N-acetylcysteine (NAC) increases intracellular stores of glutathione and has direct antioxidant properties. In this study, the effects of glutathione and NAC on the cytotoxicity of neutrophils and mononuclear cells were tested using cells from healthy controls and human immunodeficiency virus (HIV)-infected patients. NAC (1 and 5 mM) enhanced the antibody-dependent cellular cytotoxicity (ADCC) of neutrophils from healthy adult controls and HIV-infected adults and children. The antineoplastic drug, 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU), which depletes intracellular glutathione, inhibited the ADCC of neutrophils; the addition of NAC partially reversed this inhibition. Similar effects of BCNU and NAC were seen when the cytotoxicity of mononuclear cells was tested using CEM tumor cells bearing the HIV gp120 antigen as targets. Thus, NAC enhances various forms of cytotoxicity and may be beneficial to AIDS patients whose defects in leukocyte cytotoxicity may be due to glutathione depletion.

Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

Human CD4+ T Cells Can Discriminate the Molecular and Structural Context of T Epitopes of HIV gp120 and HIV p66

CD4+ T cell lines and clones specific for human immunodeficiency virus (HIV) antigens have been generated from peripheral lymphocytes of naive individuals by priming with the envelope protein gp120, the enzyme reverse transcriptase (p66), and their synthetic peptides. T cells were tested for proliferation to proteins, to peptides, and to HIV virions. Different patterns of reaction were identified. T cells primed in vitro with the whole antigen responded to the protein, but recognition of overlapping peptides occurred with a fraction of the lines or clones. The virus was recognized by some, but not all, of the gp120- and p66-specific T cells, with an efficiency 2 logs higher than the recombinant soluble proteins on a molar basis. One T cell line specific for gp120 responded to virions presented by B cells, but not by monocytes. In contrast, T cells induced with peptides did not always respond to the proteins. Generation of T cell lines from naive individuals may be an in vitro model for T cell immunization, and the response patterns may have implications for the design of vaccines aimed at inducing a T helper response. In fact our in vitro data suggest that (a) immunization with peptides does not always induce T cells recognizing the whole protein, (b) immunization with proteins does not always induce T cells recognizing the protein in the context of the HIV virus, and (c) recognition of gp120 in the context of HIV may be dictated by the type of presenting cells.

Lack of Pseudotype Formation between Human Immunodeficiency Virus Type 1 and Epstein-Barr Virus in Productively Coinfected B Lymphoblastoid Cell Lines

Human immunodeficiency virus (HIV) can form pseudotypes with other enveloped viruses, including herpes simplex virus, when the two viruses coinfect the same cell. Pseudotypes between HIV and Epstein-Barr virus (EBV) have not been described. We observed unusually high levels of HIV-1 replication in SCID mice transplanted with human peripheral blood mononuclear cells (hu-PBL-SCID mice) when the mice developed EBV-associated human B cell lymphoproliferative disease. If this enhancement of HIV-1 replication were due to pseudotype formation rather than direct infection of B lymphoblastic cells by HIV-1, the pseudotypes could pose a novel biohazard to laboratory workers. To assess whether HIV-1 and EBV can form such pseudotypes, we established and characterized CD4-positive B lymphoblastoid cell lines (LCL) that contained cells infected with both EBV and HIV-1. A high-titered virus pool from these LCL could induce HIV infection in the Burkitt's lymphoma (BL) line BJA-B, but not in the BL line Ramos. Infection of BJA-B was blocked by neutralizing antibody to HIV gp120 but not by neutralizing anti-EBV gp350. These experiments provide no evidence for pseudotype formation, suggesting a low risk for EBV:HIV pseudotypes in natural infection of humans or in human cells transplanted to SCID mice.

Generation of antigen‐specific CD4+ T cell lines from naive precursors

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (M phi). In short-term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or M phi. KLH- and SWM-specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and M phi in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.

Recombinant CD4‐IgE, a novel hybrid molecule, inducing basophils to respond to human immunodeficiency virus (HIV) and HIV‐infected target cells

Basophils and mast cells, as the main effector cells in IgE-mediated type I hypersensitivity, are involved in the elimination of parasites and, according to recent findings, may also play an important role in the defense against bacterial and viral infections. Using a genetic engineering approach we wanted to redirect this potent IgE-mediated defense system against intruding human immune deficiency virus. We constructed a recombinant CD4-IgE molecule, consisting of the two N-terminal domains of CD4 and the CH2-4 domains of the IgE heavy chain, thus providing the IgE with specificity for the gp120 of human immunodeficiency virus (HIV). The binding properties of hybrid CD4-IgE to the high-affinity receptor for IgE (Fc epsilon RI) on basophils as well as to the low-affinity receptor (Fc epsilon RII or CD23) for IgE on lymphoid cells were found to be similar to those of native IgE. At the same time, the CD4 domains of the recombinant molecule retained the gp120 binding specificity with an affinity similar to that of the native CD4. By functional tests, we demonstrated that CD4-IgE armed basophils can be triggered by free HIV and by HIV-infected cells to release their mediators. We further show that HIV-triggered basophils lead to a decreased replication of HIV in susceptible T cells. We, therefore, conclude that the type I hypersensitivity effector cells can be engaged in the elimination of HIV-infected cells, at least in vitro. Because of the strong binding of the CD4-IgE construct to the Fc epsilon RI, we assume that CD4-IgE has a short t1/2 in serum, but may similarly to IgE exhibit prolonged resident time on basophils and mast cells, which are located close to mucosal surfaces or in the connective tissue. Thus CD4-IgE could play an important role in the elimination of HIV also in vivo.

Influence of host cell type and V3 loop of the surface glycoprotein on susceptibility of human immunodeficiency virus type 1 to polyanion compounds

Dextran sulfate is a potent inhibitor of human immunodeficiency virus (HIV) binding and replication in lymphocytic cell lines. In this study, we demonstrate that the effect of dextran sulfate and heparin depends on the host cell type and on the V3 loop, the principal neutralizing determinant of HIV gp120. In particular, when dextran sulfate was tested on primary human macrophages infected with macrophage-tropic viruses, enhancement of infection was observed in 6 of 11 independent macrophage preparations and with 5 of 13 primary HIV isolates. Our in vitro observations might explain why enhanced HIV replication was observed in HIV-infected patients treated with dextran sulfate.

Expression of CD4 by human hematopoietic progenitors [see comments

It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti- CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture- initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit- erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.

Expression of CD4 by Human Hematopoietic Progenitors

It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti-CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture-initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.

Interaction of HIV gp120 and anti‐CD4 antibodies with the CD4 molecule on human CD4+ T cells inhibits the binding activity of NF‐AT, NF‐χB and AP‐1, three nuclear factors regulating interleukin‐2 gene enhancer activity

The membrane glycoprotein CD4 is required for optimal antigen-mediated activation of CD4+ T cells restricted by class II molecules of the major histocompatibility complex (MHC). CD4 cross-linking by anti-CD4 antibodies or binding by human immunodeficiency virus (HIV) gp120 has been shown to inhibit antigen-dependent and -independent T cell activation, abrogating T cell proliferation, IL-2 synthesis and the increase in the intracellular calcium concentration. The molecular basis of these opposing phenomena is ill-defined. To characterize further the inhibitory role of the CD4 molecule, we investigated the effects of CD4 ligands on the transcription factors regulating the IL-2 gene enhancer and IL-2 synthesis. We first confirmed that pre-treatment of peripheral human CD4+ T lymphocytes by CD4 ligands, HIV gp120 or anti-CD4 monoclonal antibodies inhibited IL-2 production and cell proliferation, which was normally induced by an anti-CD3 antibody (UCHT1) plus a protein kinase C activator (PMA). Moreover, these CD4 ligands inhibited the proliferation and synthesis of IL-2 induced by activators bypassing membrane events, i.e. PMA and calcium ionophore, pointing to an active signaling pathway triggered by the CD4 molecule. Gp120 and anti-CD4 antibodies induced a specific, significant decrease in the binding activity of NF-AT, NF-kappa B and AP-1, three transcription factors regulating IL-2 gene enhancer activity, as demonstrated by electrophoretic mobility shift assays. Inhibition was similarly observed following cell activation by activators involving membrane events and those bypassing them. These results strongly suggest that the inhibition mediated by cross-linking of the CD4 molecule is at least partly due to negative signal down-regulating the availability of nuclear factors necessary for the regulation of IL-2 gene transcription.

The role of immunoglobulin binding factors in the pathogenesis and therapy of AIDS

The human immunodeficiency virus (HIV) gp120 and gp41 envelope proteins and Staphylococcus aureus protein A (SPA) all have Fc receptor (FcR)-like immunoglobulin binding factor (IBF) activity for the Fc constant fragments of human immunoglobulin G (IgG). Viral IBF may contribute to the pathology of HIV by jamming the network of FcR signals that control FcR-dependent immunity. Conversely, the bacterial IBF SPA has anti-retroviral activity that may involve antagonism of the immunopathological action of viral IBF, strongly suggesting IBF may act as a double-edged sword that might be turned against viral invaders.

The role of calcium andN-linked glycans in the oligomerization and carbohydrate binding properties of human immunodeficiency virus external envelope glycoprotein

Envelope glycoproteins of human immunodeficiency virus (gp120 and gp41) occur as oligomers. Here, we show by gel filtration analysis that gp120 oligomerization in vitro is calcium- and temperature-dependent. Recombinant gp120 (rgp120) species were recovered as monomers at 20 degrees C in the absence of calcium, but as tetramers at 37 degrees C in 10 mM CaCl2. Under the latter condition, N-glycanase-deglycosylated rgp120 formed hexamers. Relative to intact rgp120, which has been reported to display carbohydrate-binding properties for N-acetyl-beta-D-glucosaminyl and mannosyl residues, deglycosylation enhanced rgp120 specific binding to mannose-divinylsulfone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and fetuin-agarose matrices. Taken together, these results rule out the role of homologous lectin-carbohydrate interactions via N-linked glycans in the rgp120 oligomerization, even though its lectin properties may also be calcium-dependent. Deglycosylation may unmask domains of rgp120 polypeptide backbone that independently play a role either in rgp120 lectin activity or in calcium-dependent oligomerization.

A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex-encoded class II molecules.

CD4, a cell-surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus-1 (HIV-1). Screening of microbial metabolites for CD4-binding activity using an enzyme-linked immunosorbent assay based on the binding of the CD4-specific monoclonal antibody (mAb), anti-Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C28 molecule probably arising from a dimerization of two C14 polyketide units. It strongly inhibited the interaction of anti-Leu 3a and that of several other D1/D2 epitope-specific mAb with CD4, but only weakly inhibited the binding of HIV-1 gp120. Binding of a representative MHC class II molecule, HLA-DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC50 = 1 microM). In functional assays 411F inhibited antigen-induced CD4-dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide-induced CD4+ T cell proliferative responses but not alloantigen-induced CD8+ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4-dependent functions by binding over a broad area of the positively charged amino-terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.

Human immunodeficiency virus-1 recombinant gp120 induces changes in protein kinase C isozymes — A preliminary report

Human immunodeficiency virus 1 (HIV-1) and its purified proteins activate target cell functions. Because protein kinase C (PKC) plays a crucial role in signal transduction and there is a molecular heterogeneity of PKC, we compared the effect of recombinant HIV-1 gp 120 and phorbol ester (PMA) on PKC isozymes in monocytic U937 cells, with isozyme-specific antibodies using flow cytometry. All PKC isozymes except PKC-γ were present in U937 cells. Both PMA and HIV-1 gp120 increased levels of calcium-dependent and -independent PKC isozymes. The most striking change was observed in PKC-ξ isozymes levels. This study for the first time demonstrate the HIV-1 gp120 affects calcium-independent PKC isozymes in U937 cells.

NMR-derived solution conformations of a hybrid synthetic peptide containing multiple epitopes of envelope protein gp120 from the RF strain of human immunodeficiency virus.

Solution conformations of a 40-residue hybrid peptide containing T-helper epitopes and B-cell determinants from envelope glycoprotein gp120 of human immunodeficiency virus (HIV) have been investigated with NMR. Peptides of this general design are highly immunogenic and induce HIV-neutralizing antibodies and T-lymphocyte responses. The 16-residue N-terminal segment of the peptide contains a T-helper epitope, while the 24-residue C-terminal segment is derived from the V3 loop of HIV strain RF and contains epitopes that elicit neutralizing antibodies as well as T-cell responses. On the basis of 2D proton NMR spectra (COSY, TOCSY, and NOESY) of the peptide in aqueous solution, the resonances of nearly all hydrogens are assigned. The peptide is largely disordered, but specific medium-range NOEs demonstrate conformational preferences in certain regions. Part of the N-terminal segment exhibits nascent helical conformation, consistent with a finding that many T-cell antigens can be modeled as amphipathic helices. In the V3-derived segment of the peptide, one region shows evidence of a tight turn conformation, corresponding to a turn found previously in V3 peptides of HIV strains MN and IIIB. Other conformational features are also detected in the V3 region, such as a stretch of beta strand and a kink that may arise from side-chain interactions.

Design and Construction of Rhinovirus Chimeras Incorporating Immunogens from Polio, Influenza, and Human Immunodeficiency Viruses

This paper describes the design and construction of chimeric human rhinoviruses that contain immunogenic regions from other pathogens as part of their surface coat proteins. Segments encoding the poliovirus 3 Sabin VP1 and VP2 proteins, the influenza hemagglutinin (HA) glycoprotein, and the human immunodeficiency virus gp120 surface and gp41 transmembrane glycoproteins were inserted into a full-length clone of human rhinovirus 14 (HRV14) at regions corresponding to neutralizing immunogenic sites IA (NIm-IA) and II (NIm-II). Of 12 chimeric constructs described, 3 produced viable virus. An HRV14 chimeric virus containing five amino acids of influenza HA (corresponding to 300 Å 2 of solvent-accessible surface area) had wild-type HRV14 growth characteristics and was neutralized by four of four anti-influenza HA antisera with reciprocal neutralizing titers ranging from 180 to 330. However, antisera raised in two guinea pigs against the HRV14:influenza HA chimera did not show significant neutralization of relevant strains of influenza. These results are the first to demonstrate the feasibility of making viable chimeras of human rhinoviruses displaying heterologous immunogens.

Selective effects of human immunodeficiency virus (HIV) gp120 on invertebrate neurons

1. HIV gp120 selectively reduces the glutamate-induced inward current and the acetylcholine-induced outward current in specific and identified Aplysia neurons without affecting dopamine (DA)- and serotonin (5-HT)-induced responses. 2. gp120 specifically decreases DA levels without significantly altering norepinephrine and 5-HT levels in Mytilus pedal ganglia. 3. The gp120-associated decrease in DA levels in Mytilus is dose dependent and exhibits a threshold level. 4. The alteration of in vitro DA levels is specific for gp120 since anti-gp120 blocks the effect. 5. gp120 and its effects appear to be stable due to the duration of treatment and the failure of secondary effects to materialize following antibody treatment.