Lipid conjugates of antiretroviral agents. I. Azidothymidine-monophosphate-diglyceride: anti-HIV activity, physical properties, and interaction with plasma proteins

3'-Azido-3'-deoxythymidine-5'-phosphate diglyceride (16:0/18:1 omega 9), a phosphatic acid conjugate of AZT, is active against HIV replication in H9 cells and syncytia formation in MOLT-3 cells. The activities rank as AZT greater than pure conjugate greater than conjugate in mixed liposomes, with the pure conjugate having about one-third the activity of free AZT. The compound binds very rapidly to serum lipoproteins, but not to serum albumin, alpha and beta globulins, or red cells. Pancreatic phospholipase A2 hydrolyzes it to the lysophosphatidic acid conjugate.     

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.     

Efficacy and safety of foscarnet for recurrent orolabial herpes: a multicentre randomized double-blind study.

Foscarnet sodium (trisodium phosphonoformate hexahydrate) has been shown to inhibit herpes simplex virus (HSV) in vitro and to be efficacious for topical treatment of experimental HSV infection in animals. To assess its clinical efficacy in the treatment of recurrent orolabial herpes a multicentre collaborative, double-blind, placebo-controlled trial was conducted. The study patients were randomly assigned to receive either 3% foscarnet cream (78 patients) or placebo (cream vehicle) (75 patients) and were asked to start treatment at the earliest indication of a recurrence. Efficacy was evaluated in 143 patients (74 in the foscarnet group and 69 in the placebo group). There was no significant difference in time to healing or duration of virus shedding between the two groups. However, in the subgroup of patients who started treatment before vesicles appeared, the duration of virus shedding was shorter in the foscarnet group than in the placebo group (p = 0.04), and the proportion of lesions that evolved to the vesicular stage was smaller (p = 0.03). No significant difference in the incidence of local or systemic adverse effects was noted between the two groups. We conclude that the beneficial effect of foscarnet was limited to a subgroup of patients who started treatment in the prevesicular stage.     

The human villous cytotrophoblast: Interactions with extracellular matrix proteins, endocrine function, and cytoplasmic differentiation in the absence of syncytium formation

Human syncytiotrophoblasts are derived from villous cytotrophoblasts by cell fusion. Coincident with this morphologic transformation, trophoblasts acquire specific endocrine functions, including elaboration of chorionic gonadotropin (hCG). We wondered if syncytia formation was a prerequisite for biochemical differentiation or simply was one part of the differentiation program. By growing purified human cytotrophoblasts under serum-free conditions and manipulating the culture surface, we were able to disassociate morphologic from biochemical differentiation. We have shown previously (Endocrinology 1986, 118:1567) that human cytotrophoblasts grown in the presence of fetal calf serum flatten out, aggregate, and form functional syncytiotrophoblasts in vitro over 24-96 hr. Here we demonstrate that when grown in the absence of serum, the cells do not undergo these morphologic changes, but remain as individual spherical cells. If the culture surface was precoated with fibronectin or a variety of collagens, but not albumin, the cells regained their ability to flatten, aggregate, and form syncytia. Attachment to and syncytia formation on fibronectin was blocked by the addition of the R-G-D-S-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro. Attachment to and syncytia formation on type I collagen was blocked by anti-human fibronectin F(ab')2 fragments, while association with type IV collagen was not affected by this antibody, suggesting that fibronectin mediates trophoblast association with type I collagen, but not type IV. Although syncytia formation did not occur when cytotrophoblasts were cultured under serum-free conditions in the absence of ECM proteins, biochemical differentiation was not affected. These cells secreted hCG at the same rate under serum-free conditions whether they were plated on plastic only--which prevented syncytia formation--or fibronectin, laminin or, type IV collagen--which allowed syncytia formation to occur. Furthermore, cytoplasmic differentiation in the absence of syncytia formation was confirmed by performing transmission electron microscopy on cytotrophoblasts grown under serum-free conditions in the presence of 8-bromo-cAMP. We conclude that syncytia formation is not a prerequisite for biochemical differentiation, but simply part of the trophoblast differentiation program.     

Imaging-based assay for identification and characterization of inhibitors of CXCR4-tropic HIV-1 envelope-dependent cell-cell fusion

Infection of certain cell types by HIV results in formation of syncytia. This process can be blocked by antibodies or compounds that prevent interaction of viral envelope protein with host cell receptors. Here the authors describe an automated imaging-based assay for inhibitors of cell-cell fusion mediated by interaction of HIV gp120 with CXCR4 coreceptor. The assay quantifies syncytia formation between U87MG astrocytoma cells constitutively expressing CD4/CXCR4 and morphologically distinct Jurkat T lymphoma cells inducibly expressing HIV env. Each cell type was differentially labeled with vital dyes. Fusion was quantified by measuring size, shape, and color of Jurkat cells and Jurkat-harboring cell syncytia. Dose-response experiments with reference inhibitors AMD 3100 and KRH-1636 yielded potencies consistent with those obtained using standard antiviral assays. This assay complements virus-based infectivity assays for identification of inhibitors of membrane fusion events triggered by interaction of HIV gp120 with host CXCR4.     

Fusion from without directed by human immunodeficiency virus particles.

Fusion from without is the process through which particles of some enveloped viruses can direct fusion of target cells in the absence of viral replication. We demonstrate here that human immunodeficiency virus (HIV) particles can efficiently promote fusion from without. Using HeLa-CD4 cells carrying a Tat-inducible lacZ gene, we observed syncytia as early as 6 h after exposure to HIV particles, before HIV gene expression could be detected. Efficient syncytium formation could be obtained when cells were treated with zidovudine, which prevented HIV replication and expression but not cell-cell fusion. Fusion was also observed when cells were exposed to particles of a replication-defective HIV integrase mutant. Fusion from without by HIV particles could be blocked by a monoclonal antibody specific for the V3 loop of the HIV-1 envelope glycoprotein and by soluble CD4. This mechanism of cytopathicity, which can involve cells that do not actively replicate HIV and can be directed by replication-defective particles, could participate in the pathogenicity of the CD4 cell depletion that characterizes HIV infection.     

Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.     

AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency

Acquired immune deficiency syndrome (AIDS) is a disease caused by the human immunodeficiency virus (HIV) in which cellular immune functions are severely impaired. Acute infection and subsequent destruction of helper T cells, although occurring readily in cell cultures, do not appear to be the only mechanisms mediating helper T cell loss. Other mechanisms that may account for the loss of helper T cells include: T cell syncytia formation, decreased T cell production, and autoimmune-related destruction of helper T cells. Immune abnormalities seen early in the course of HIV infection include immune hyperactivation and autoimmune phenomena suggestive of immune dysregulation rather than immune deficiency. Many changes in immune function are, in fact, seen in HIV-seropositive patients who possess a normal number of helper T cells. Mechanisms (other than the loss of helper T cells) that may contribute to the immune abnormalities seen in these patients include noninfectious effects of HIV and HIV proteins, effects of HIV on non-T cells, autoimmune-related manifestations of HIV infection, and HIV-induced activation of normal immunosuppressive circuits.     

A major mechanism of human immunodeficiency virus-induced cell killing does not involve cell fusion.

In vitro studies indicate that human immunodeficiency virus (HIV) infections are cytopathic for T4+ peripheral blood lymphocytes and for most continuous lines of T4+ lymphocytes. These cytopathic effects have been largely attributed to the formation of syncytia by HIV-infected cells. We report that HIV infections killed cultured peripheral blood lymphocytes and a line of T4+-lymphoid cells (CEM cells) without causing cell fusion. We also report that the occurrence of syncytia is an early and transitory phenomenon following infection of a fusion-susceptible line of T4+-cells (H9 cells). Mixing experiments and flow cytometry have been used to demonstrate that susceptibility to HIV-induced fusion is not determined by differences in presentation of viral envelope antigens or the surface levels of T4 receptor antigens on fusion-susceptible and -resistant cells. We conclude that a major mechanism of HIV-induced cell killing does not involve cell fusion and that HIV-induced cell fusion, when it does occur, requires factors in addition to viral envelope antigens and host T4 receptors.     

Role of the fusogenic peptide sequence in syncytium induction and infectivity of human immunodeficiency virus type 2.

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.     

Early activation events render T cells susceptible to HIV-1-induced syncytia formation. Role of protein kinase C.

In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.     

Human immunodeficiency virus envelope glycoprotein/CD4-mediated fusion of nonprimate cells with human cells.

Human immunodeficiency virus (HIV) infects human cells by binding to surface CD4 molecules and directly fusing with the cell membrane. Although mouse cells expressing human CD4 bind HIV, they do not become infected, apparently because of a block in membrane fusion. To study this problem, we constructed a recombinant vaccinia virus that can infect and promote transient expression of full-length CD4 in mammalian cells. This virus, together with another vaccinia recombinant encoding biologically active HIV envelope glycoprotein gp160, allowed us to study CD4/gp160-mediated cell-cell fusion in a wide variety of human and nonhuman cells in the absence of other HIV proteins. By using syncytium formation assays in which a single cell type expressed both CD4 and gp160, we demonstrated membrane fusion in lymphoid and nonlymphoid human cells but not in any of the 23 tested nonhuman cell types, derived from African green monkey, baboon, rabbit, hamster, rat, or mouse. However, in mixing experiments with one cell type expressing CD4 and the other cell type expressing gp160, all of these nonhuman cells could form CD4/gp160-mediated syncytia when mixed with human cells; in 20 of 23 cases, membrane fusion occurred only if the CD4 molecule was expressed on the human cells whereas in the other three cases, CD4 could be expressed on either one of the fusing partners. Interestingly, in one mouse cell line, CD4-dependent syncytia formed without a human partner, but only if a C-terminally truncated form of the HIV envelope glycoprotein was employed. Our results indicate that nonhuman cells are intrinsically capable of undergoing CD4/gp160-mediated membrane fusion, but this fusion is usually prevented by the lack of helper or the presence of inhibitory factors in the nonhuman cell membranes.     

Determination of phosphonoformate (foscarnet) in biological fluids by ion-pair reversed-phase liquid chromatography

Bioanalytical liquid chromatographic methods for the determination of phosphonoformate (foscarnet) have been developed. Biological fluids, after simple pre-treatment (ultrafiltration and/or treatment with charcoal), were injected into a reversed-phase liquid chromatographic system with electrochemical detection. Foscarnet was retained as an ion pair with tetrahexylammonium; addition of pyrophosphate was necessary in order to obtain an acceptable peak. This additive could also be used for the fine regulation of the retention to achieve the necessary selectivity.     

Induction of syncytia by Moloney murine leukemia virus in myoblasts defective in differentiation.

fu-1 cells, a nonfusing variant of the L8 line of rat myoblasts, form syncytia upon infection with murine leukemia virus (MuLV) or upon cocultivation with MuLV-infected cells; L8 cells do not form these syncytia, but do fuse into multinucleate myotubes. Syncytia of fu-1 cells form within 1 h after infection. The number of syncytia formed is proportional to the multiplicity of virus within a range of 4 to 16 and is maximum when the cell density is subconfluent. When either XC or fu-1 cells are productively infected with MuLV, they become resistant to syncytia formation by passage 3. The rapid formation of syncytia in fu-1 cells was found amenable for selection of temperature-sensitive mutants of MuLV and for screening additional variants of the L8 line.     

Glycosylphosphatidylinositol-anchored CD4 supports human immunodeficiency virus type 1 replication, but not cytopathic effect, in T-cell transfectants.

Despite equivalent p24 antigen production, HSB-2 T cells expressing glycosylphosphatidylinositol (GPi)-linked CD4 were productively infected without cell death or syncytium formation, unlike HSB-2 transfectants expressing wild-type CD4 (wtCD4). HSB-2 transfectants dually expressing wtCD4 and GPi-linked CD4 formed syncytia and died. Thus, wtCD4 expression is critical for human immunodeficiency virus cytopathic effect in HSB-2 transfectants.     

Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.

We generated Chinese hamster ovary cell lines that stably express wild-type, secreted, and glycosylphosphatidylinositol (GPI)-anchored envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The cells expressing wild-type Env (WT cells) express both the precursor gp160 and the mature gp120/gp41 and readily form large syncytia when cocultivated with CD4+ human cells. The cells expressing secreted Env (SEC cells) release 140-kDa precursor and mature 120-kDa envelope glycoproteins into the supernatants. The cells expressing GPI-anchored Env (PI cells) express both 140-kDa precursor and mature gp120/gp41 envelope glycoproteins, which can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Both the secreted and PI-PLC-released envelope glycoproteins form oligomers that can be detected on nonreducing sodium dodecyl sulfate-polyacrylamide gels. In contrast to the WT cells, the SEC and PI cells do not form syncytia when cocultivated with CD4+ human cells. The availability of cells producing water-soluble oligomers of HIV-1 Env should facilitate studies of envelope glycoprotein structure and function. The WT cells, which readily induce syncytia with CD4+ cells, provide a convenient system for assessing potential fusion inhibitors and for studying the fusion mechanism of the HIV Env glycoprotein.     

Electron microscopic study of measles virus infection: cell fusion and hemadsorption.

Virus-induced cell fusion has been studied after infection of Vero cells with measles virus. Scanning and transmission electron microscopy were combined with immunoperoxidase labeling of measles antigens to correlate viral production and distribution of virus-induced erythrocyte binding sites with progress of fusion. Release of infectious virus started before syncytia were detected and decreased while the number and size of syncytia were increasing. Most virions were seen budding from mononucleated cells or from the periphery of syncytia where cells were being recruited. Moving inward, the surfaces of syncytia where cells were being recruited. Moving inward, the surfaces of syncytia were covered with numerous ridges containing viral antigen, but few viral buds were seen, suggesting that syncytia might be sites of defective viral formation. Hemadsorption occurred predominantly within the confines of syncytia. Erythrocytes were scattered sparsely over immature syncytia but were densely packed in the center of mature syncytia. Active binding sites for erythrocytes were located on cell villi and ridges covered with measles antigens. Hemadsorption was completely inhibited in measles virus-infected cultures pretreated with virus-specific immunoglobulin G for 1 h at 4 degrees C. However, when these cultures were shifted to 37 degrees C, hemadsorbing sites were recovered at the periphery of enlarging syncytia. Virus-induced sites for erythrocyte adsorption were found to move centripetally on syncytium membranes as fusion progressed.