Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.     

Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding

The human immunodeficiency virus (HIV) binds to the surface of T lymphocytes and other cells of the immune system via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gp120. By analogy with certain other enveloped viruses, receptor binding by HIV may be followed by exposure of the hydrophobic NH2 terminus of its transmembrane glycoprotein, gp41, and fusion of the virus and cell membranes. A similar sequence of events is thought to take place between HIV-infected and uninfected CD4+ cells, resulting in their fusion to form syncytia. In this study, we have used a soluble, recombinant form of CD4 (sCD4) to model events taking place after receptor binding by the HIV envelope glycoproteins. We demonstrate that the complexing of sCD4 with gp120 induces conformational changes within envelope glycoprotein oligomers. This was measured on HIV-1-infected cells by the increased binding of antibodies to the gp120/V3 loops, and on the surface of virions by increased cleavage of this loop by an exogenous proteinase. At 37 degrees C, these conformational changes are coordinate with the dissociation of gp120/sCD4 complexes from gp41, and the increased exposure of gp41 epitopes. At 4 degrees C, gp120 dissociation from the cell surface does not occur, but increased exposure of both gp120/V3 and gp41 epitopes is detected. We propose that these events occurring after CD4 binding are integral components of the membrane fusion reaction between HIV or HIV-infected cells and CD4+ cells.     

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.     

Anti-HIV-1 activity of enfuvirtide (T-20) by inhibition of bystander cell death

Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death of both infected and bystander cells. The envelope glycoprotein complex appears to play an active role in HIV-induced death of bystander cells. We quantified cell-to-cell fusion, single cell death and membrane lipid mixing in cocultures of effector, HIV-1 envelope-expressing cells with peripheral blood mononuclear cells or purified CD4 T lymphocytes from HIV-negative donors, in the presence or the absence of the fusion inhibitor enfuvirtide (T-20, pentafuside, Fuzeon). T-20, which blocks gp41-dependent virus-cell fusion, showed a complete and dose-dependent inhibition of syncytium formation in cocultures of envelope-expressing cells with uninfected cells. Similarly, T-20 totally abrogated death of single bystander CD4 T cells with an IC50 of 0.04 microg/ml. Membrane lipid mixing, as a measure of interaction between envelope-expressing cells and CD4 cells, was also dose-dependently inhibited by T-20. Moreover, effector cells chronically infected with a T-20-resistant virus recovered the ability to induce bystander cell death in the presence of the drug, supporting the role of gp41 in single cell death. In conclusion, T-20 is able to protect CD4 T cells from envelope presentation with a dual effect: inhibition of virus replication and blockade of HIV-1 envelope-induced cell death of bystander CD4 T cells. Protection of cells prior to infection from HIV envelope-dependent bystander effect could lead to a better immune restoration of HIV-1-infected patients that are treated with T-20.     

Role of human immunodeficiency virus (HIV) type 1 envelope in the anti-HIV activity of the betulinic acid derivative IC9564

The betulinic acid derivative IC9564 is a potent anti-human immunodeficiency virus (anti-HIV) compound that can inhibit both HIV primary isolates and laboratory-adapted strains. However, this compound did not affect the replication of simian immunodeficiency virus and respiratory syncytial virus. Results from a syncytium formation assay indicated that IC9564 blocked HIV type 1 (HIV-1) envelope-mediated membrane fusion. Analysis of a chimeric virus derived from exchanging envelope regions between IC9564-sensitive and IC9564-resistant viruses indicated that regions within gp120 and the N-terminal 25 amino acids (fusion domain) of gp41 are key determinants for the drug sensitivity. By developing a drug-resistant mutant from the NL4-3 virus, two mutations were found within the gp120 region and one was found within the gp41 region. The mutations are G237R and R252K in gp120 and R533A in the fusion domain of gp41. The mutations were reintroduced into the NL4-3 envelope and analyzed for their role in IC9564 resistance. Both of the gp120 mutations contributed to the drug sensitivity. On the contrary, the gp41 mutation (R533A) did not appear to affect the IC9564 sensitivity. These results suggest that HIV-1 gp120 plays a key role in the anti-HIV-1 activity of IC9564.     

Processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin

The processing and secretion of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected cells treated with the trimming glucosidase inhibitor deoxynojirimycin (DNM). In Molt3 cells infected with human T-lymphotropic virus type III (HTLV-IIIB), DNM inhibited the intracellular proteolytic processing of gp160 to gp120 and gp41. A clone of the HUT78 cell line called 6D5, when chronically infected with the HIV-1 isolate HTLV-III451 was shown to release both gp160 and gp120 into the culture medium. The secretion of envelope glycoproteins from these infected cells was not inhibited by DNM treatment. The secreted proteins had higher molecular weights than gp160 and gp120 from cultures not treated with DNM, presumably due to the presence of unprocessed carbohydrate residues on the polypeptide chain. These secreted glycoproteins from DNM-treated cells exhibited specific interaction with the CD4 molecule on the surface of target cells. However, the syncytium formation induced by HIV-1-infected cells on CD4+ cells was significantly inhibited in the presence of the glucosidase inhibitor. The minimal cytotoxicity of the DNM coupled with its strong inhibitory effect on the cell-to-cell spread of the virus suggest that it may be potentially useful in antiviral drug therapy of HIV-1 infection.     

HIV-1 proprotein processing as a target for gene therapy

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.     

Structure and function of oligosaccharide chains of the envelope glycoprotein gp120 of human immunodeficiency virus type 1]

The surface of the human immunodeficiency virus (HIV-1), a causative agent for acquired immunodeficiency syndrome (AIDS), is covered with the major envelope glycoprotein gp120, of which the carbohydrate moiety accounted for 50% of the molecular mass. There is evidence that glycosylation of gp120 is prerequisite to the various stages of HIV infection. The oligosaccharide structures of gp120 have been determined using recombinant gp120 of HIV-1 (IIIB) produced in chinese hamster ovary cells and virus-derived gp120 isolated from H9 lymphocytes chronically infected with HIV-1 (IIIB). Three oligosaccharides have been suggested to be involved in the HIV-infection process. Occurrence of infection process which is independent of CD4 recognition and mediated by gp120 oligosaccharides, mannose-binding protein, and complement system has been suggested.     

Technology evaluation: HIVAC-1e

Bristol-Myers Squibb is developing a vaccine, HIVAC-1e, comprised of a recombinant vaccinia virus expressing the HIV-1 gp160 envelope glycoprotein. The vaccine has potential for the treatment of HIV and other viral infections and has entered phase I trials [135333]. In an initial phase I trial, 11 vaccinia-na?ve volunteers were vaccinated with HIVAC-1e, followed by a booster with baculovirus-derived gp160 (VaxSyn). Two weeks after boosting, all 11 volunteers developed HIV-1 specific IgG, with titers of 1:40 to 1:1280 [195287]. Using the same strategy in 29 vaccinia-na?ve volunteers, priming with HIVAC-1e was demonstrated as a key determinant of the epitope specificity and magnitude of antibody responses to gp160 [195278].     

HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse

Direct cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4(+)/CXCR4(+) target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.     

Inhibition of coreceptor-independent cell-to-cell human immunodeficiency virus type 1 transmission by a CD4-immunoglobulin G2 fusion protein

We have previously shown (J. Blanco et al., J. Biol. Chem. 279:51305-51314, 2004) that the contact between HIV producing cells and primary CD4(+) T cells may induce the uptake of human immunodeficiency virus (HIV) particles by target cells in the absence of HIV envelope-mediated membrane fusion or productive HIV replication. HIV uptake by CD4(+) T cells was dependent on cellular contacts mediated by the binding of gp120 to CD4 but was independent of the expression of the appropriate HIV coreceptor, CCR5 or CXCR4. Here, we have characterized the effect of agents blocking gp120 binding to CD4 on cell-to-cell HIV transmission. A recombinant CD4-based protein (CD4-immunoglobulin G2 [IgG2]), that is currently being evaluated in clinical trials, completely inhibited the uptake of HIV particles by CD4(+) T cells from persistently infected cells expressing R5, X4, or X4/T-20-resistant HIV-1 envelope glycoproteins. Consequently, both the release of viral particles from endocytic vesicles and the infection of reporter U87-CD4 cells were also prevented. The polyanionic anti-HIV agent dextran sulfate failed to prevent the intracellular uptake of virions by CD4(+) T cells. Indeed, it increased HIV uptake in a dose-dependent manner, suggesting functional differences between the specific gp120-targeting CD4-IgG2 agent and nonspecific HIV binding inhibitors. Thus, the inhibition of the specific interaction between gp120 and CD4 protein could be an effective strategy to inhibit HIV binding to CD4(+) T cells, and the mechanism by which CD4(+) T cells lacking the appropriate coreceptor may be converted in HIV carriers.     

The Phthalocyanine Prototype Derivative Alcian Blue Is the First Synthetic Agent with Selective Anti-Human Immunodeficiency Virus Activity Due to Its gp120 Glycan-Binding Potential

Alcian Blue (AB), a phthalocyanine derivative, is able to prevent infection by a wide spectrum of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains in various cell types [T cells, (co)receptor-transfected cells, and peripheral blood mononuclear cells]. With the exception of herpes simplex virus, AB is inactive against a broad variety of other (DNA and RNA) viruses. Time-of-addition studies show that AB prevents HIV-1 infection at the virus entry stage, exactly at the same time as carbohydrate-binding agents do. AB also efficiently prevents fusion between persistently HIV-1-infected HUT-78 cells and uninfected (CD4⁺) lymphocytes, DC-SIGN-directed HIV-1 capture, and subsequent transmission to uninfected (CD4⁺) T lymphocytes. Prolonged passaging of HIV-1 at dose-escalating concentrations of AB resulted in the selection of mutant virus strains in which several N-glycans of the HIV-1 gp120 envelope were deleted and in which positively charged amino acid mutations in both gp120 and gp41 appeared. A mutant virus strain in which four N-glycans were deleted showed a 10-fold decrease in sensitivity to the inhibitory effect of AB. These data suggest that AB is likely endowed with carbohydrate-binding properties and can be considered an important lead compound in the development of novel synthetic nonpeptidic antiviral drugs targeting the glycans of the envelope of HIV.Targeting the entry process of human immunodeficiency virus (HIV), including drugs that bind to the receptor CD4, to a coreceptor, CCR5 or CXCR4, or to gp160 envelope is a valuable approach to prevent or suppress HIV infections. The first clinically used entry inhibitor, enfuvirtide (T20; Fuzeon) binds to the transmembrane gp41 of the envelope of HIV (21), thus preventing the required conformational changes of the envelope to successfully complete viral entry. Most recently, the CCR5 antagonist maraviroc was approved by the FDA for the treatment of HIV-infected individuals (16). Both enfuvirtide and maraviroc prove that HIV entry can be efficiently targeted by drugs that act at different stages in the entry process.The envelope of HIV consists of two subunits: the surface gp120 and the transmembrane gp41. Both units are highly glycosylated (23, 24), which is essential for the virus to escape immune surveillance (28). A broad variety of carbohydrate-binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), and Urtica dioica agglutinin (UDA) or the prokaryotic cyanovirin-N (CV-N) and actinohivin, bind to the glycans that are present on the envelope of HIV and inhibit the viral entry process (4, 5). The majority of the natural CBAs are proteins (i.e., lectins), which may have some major drawbacks: (i) it is technically not easy and it is relatively costly to produce and purify these proteins on a large scale, (ii) they have poor, if any, oral bioavailability, and (iii) they can trigger an immune response that compromises their eventual antiviral efficacy (1). Therefore, (semi)synthetic low-molecular-weight compounds that are also able to bind glycans and prevent virus infection would be a valuable alternative.Recently, pradimicin A (PRM-A), an antifungal nonpeptidic antibiotic (26), was described to possess lectin-like properties and bind to the glycans of HIV gp120 (32) and proved able to efficiently prevent HIV infection (31). HIV selected under escalating PRM-A concentrations can escape this drug pressure by deleting multiple N-glycosylation sites in gp120 (10). This was earlier also observed to occur in HIV-1 strains selected under pressure of peptidic CBAs, such as HHA, GNA, UDA, and cyanovirin-N (5, 6). Thus, the CBAs not only prevent virus infection by binding to glycans on the envelope of HIV, but also they can force the virus to delete its envelope N-glycans in order to escape drug pressure, resulting in the exposure of previously hidden immunogenic epitopes. This phenomenon is interesting given the fact that the glycans on the HIV gp120 envelope play a very important role in shielding the virus from the immune system and in limiting the neutralizing antibody response to HIV (39).Here, we report on a synthetic compound, the phthalocyanine Alcian Blue (AB), that is endowed with anti-HIV activity due to its lectin-like properties. It prevents entry of HIV into its target cells and selects for mutant virus strains that have several deletions in N-glycosylation sites in gp120.     

Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.     

Sequential immunizations with rgp120s from independent isolates of human immunodeficiency virus type 1 induce the preferential expansion of broadly crossreactive B cells

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is a dominant target against which the host's humoral immune response is directed. Unfortunately, gp120 proteins from different isolates of HIV are antigenically distinct, complicating the use of the envelope glycoprotein in vaccines designed to prevent acquired immunodeficiency syndrome. Using an enzyme-linked immunosorbent spot assay (ELISA), BALB/c mice immunized and boosted with recombinant purified gp120 were studied at the single cell level for their humoral immune response to HIV-1 envelope proteins. Approximately 90% of responding B cells produced antibodies reactive with the immunizing form of gp120 but not with gp120s from other strains of HIV. A novel sandwich ELISA was then used to analyze the frequency with which individual in vivo activated B cells produced antibodies that crossreacted with heterologous gp120s. Repeated immunizations with a single gp120 or with a mixture of different gp120s resulted in the activation of primarily mono-specific (noncrossreactive) B cells. In contrast, the sequential immunization of mice with recombinant purified envelope proteins from different strains of HIV (IIIB, SF2, and Zr6) induced the selective expansion of B cells producing highly crossreactive antibodies.     

Apolipoprotein A-I and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation.

The envelope (membrane) glycoprotein of HIV is essential for virus attachment and entry into host cells. Additionally, when expressed on the plasma membrane of infected cells, the envelope protein is responsible for mediating cell-cell fusion which leads to the formation of multinucleated giant cells, one of the major cytopathic effects of HIV infections. The envelope glycoproteins of HIV contain regions that can fold into amphipathic alpha-helixes, and these regions have been suggested to play a role in subunit associations and in virus-induced cell fusion and cytopathic effects of HIV. We therefore tested the possibility that amphipathic helix-containing peptides and proteins may interfere with the HIV amphipathic peptides and inhibit those steps of HIV infection involving membrane fusion. Apolipoprotein A-I, the major protein component of high density lipoprotein, and its amphipathic peptide analogue were found to inhibit cell fusion, both in HIV-1-infected T cells and in recombinant vaccinia-virus-infected CD4+ HeLa cells expressing HIV envelope protein on their surfaces. The amphipathic peptides inhibited the infectivity of HIV-1. The inhibitory effects were manifest when the virus, but not cells, was pretreated with the peptides. Also, a reduction in HIV-induced cell killing was observed when virus-infected cell cultures were maintained in presence of amphipathic peptides. These results have potential implications for HIV biology and therapy.     

CD4-Pseudomonas exotoxin conjugates delay but do not fully inhibit human immunodeficiency virus replication in lymphocytes in vitro.

The CD4 molecule is a high affinity receptor for the human immunodeficiency virus (HIV) envelope glycoprotein (gp160 or gp120). This glycoprotein is expressed on the surface membrane of cells infected with HIV. It has, therefore, been suggested that a soluble form of CD4 might be used as a targeting agent to deliver toxins selectively to cells infected with HIV. We demonstrate that CD4-Pseudomonas exotoxin A (PE) conjugates inhibit the proliferation of gp160-transfected Chinese hamster ovary cells and block HIV replication in virus-infected H9 cells. However, this inhibition of HIV replication appears to be incomplete since virus replication occurs following removal of the toxin conjugates from these cultures. Moreover, CD4-PE conjugates delay but do not inhibit HIV replication in human peripheral blood lymphocytes. These studies suggest that such conjugates should be assessed only as potential adjunctive therapies in the acquired immunodeficiency syndrome.