Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation

Cell fusion (syncytium formation) is a major cytopathic effect of infection by human immunodeficiency virus (HIV) and may also represent an important mechanism of CD4+ T-cell depletion in individuals infected with HIV. Syncytium formation requires the interaction of CD4 on the surface of uninfected cells with HIV envelope glycoprotein gp120 expressed on HIV-infected cells. However, several observations suggest that molecules other than CD4 play a role in HIV-induced cell fusion. The leukocyte adhesion receptor LFA-1 is involved in a broad range of leukocyte interactions mediated by diverse receptor-ligand systems including CD4-class II major histocompatibility complex (MHC) molecules. Possible mimicry of class II MHC molecules by gp120 in its interaction with CD4 prompted an examination of the role of LFA-1 in HIV-induced cell fusion. A monoclonal antibody against LFA-1 completely inhibited HIV-induced syncytium formation. The antibody did not block binding of gp120 to CD4. This demonstrates that a molecule other than CD4 is also involved in cell fusion mediated by HIV.     

Epitopes of the CD4 antigen and HIV infection

The CD4 (or T4) surface antigen of human T lymphocytes is an important part of the receptor for the human immunodeficiency virus (HIV). After binding to the receptor, the HIV may enter the T cell and induce the formation of syncytia. In an attempt to identify the receptor site more closely, monoclonal antibodies (Mab's) to CD4 were tested for their ability to block HIV infection in a syncytium formation assay, and the CD4 epitopes so identified were mapped by antibody cross-blocking. The antibodies that showed strong inhibition of HIV fell into two main families while a third group of Mab's blocked syncytia formation weakly or not at all. Several different isolates of HIV as well as the laboratory strain CBL1 grown in CEM cells were used to induce the syncytia. The data indicate that only some epitopes of CD4 are important for virus binding and imply that the virus-binding site for CD4 is conserved in different isolates of HIV with substantially divergent env gene sequences. Preliminary studies of patients suggest that polymorphism of these epitopes does not play a role in determining susceptibility to infection.     

Use of CD134 as a primary receptor by the feline immunodeficiency virus

Feline immunodeficiency virus (FIV) induces a disease similar to acquired immunodeficiency syndrome (AIDS) in cats, yet in contrast to human immunodeficiency virus (HIV), CD4 is not the viral receptor. We identified a primary receptor for FIV as CD134 (OX40), a T cell activation antigen and costimulatory molecule. CD134 expression promotes viral binding and renders cells permissive for viral entry, productive infection, and syncytium formation. Infection is CXCR4-dependent, analogous to infection with X4 strains of HIV. Thus, despite the evolutionary divergence of the feline and human lentiviruses, both viruses use receptors that target the virus to a subset of cells that are pivotal to the acquired immune response.     

Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1

The envelope of the human immunodeficiency virus type 1 (HIV-1) plays a central role in the process of virus entry into the host cell and in the cytopathicity of the virus for lymphocytes bearing the CD4 molecule. Mutations that affect the ability of the envelope glycoprotein to form syncytia in CD4+ cells can be divided into five groups: those that decrease the binding of the envelope protein to the CD4 molecule, those that prevent a post-binding fusion reaction, those that disrupt the anchorage of the envelope glycoprotein in the membrane, those that affect the association of the two subunits of the envelope glycoprotein, and those that affect post-translational proteolytic processing of the envelope precursor protein. These findings provide a functional model of the HIV envelope glycoprotein.     

Synthetic CD4 peptide derivatives that inhibit HIV infection and cytopathicity

Synthetic peptide segments of the CD4 molecule were tested for their ability to inhibit infection of CD4+ cells by the human immunodeficiency virus (HIV) and to inhibit HIV-induced cell fusion. A peptide mixture composed of CD4(76-94), and synthesis side products, blocked HIV-induced cell fusion at a nominal concentration of 125 micromolar. Upon high-performance liquid chromatography, the antisyncytial activity of the peptide mixture was found not in the fraction containing the peptide CD4(76-94) itself, but in a side fraction containing derivatized peptide products generated in the automated synthesis. Derivatized deletion and substitution peptides in the region CD4(76-94) were used to demonstrate sequence specificity, a requirement for benzyl derivatization, and a core seven-residue fragment required for antisyncytial activity. A partially purified S-benzyl-CD4(83-94) peptide mixture inhibited HIV-induced cell fusion at a nominal concentration of less than or equal to 32 micromolar. Derivatized CD4 peptides blocked cell fusion induced by several HIV isolates and by the simian immunodeficiency virus, SIV, and blocked infection in vitro by four HIV-1 isolates with widely variant envelope gene sequences. Purified CD4(83-94) dibenzylated at cysteine 86 and glutamate 87 possessed antisyncytial activity at 125 micromolar. Derivatization may specifically alter the conformation of CD4 holoreceptor peptide fragments, increasing their antiviral efficacy.     

Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization

Rabbit antisera were raised against three overlapping synthetic peptides with sequence homology to the second conserved domain of the external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV). All of the antisera immunoprecipitated the envelope glycoprotein. In particular, an antiserum directed against amino acids 254 to 274 of env was efficient in neutralizing three different isolates of HIV in vitro, without affecting the binding of the virus to CD4-positive cells. Therefore, this conserved region of gp120 appears to be critical in a postbinding event during virus penetration and may represent a target for antibody neutralization of HIV. These findings may be applicable in the design of a vaccine for the acquired immunodeficiency syndrome.     

Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide.

Although the CD4 molecule is the principal cellular receptor for the human immunodeficiency virus (HIV), several CD4-negative cell lines are susceptible to infection with one or more HIV strains. These findings indicate that there are alternate modes of viral entry, perhaps involving one or more receptor molecules. Antibodies against galactosyl ceramide (galactocerebroside, or GalC) inhibited viral internalization and infection in two CD4-negative cell lines derived from the nervous system: U373-MG and SK-N-MC. Furthermore, recombinant HIV surface glycoprotein gp120 bound to GalC but not to other glycolipids. These results suggest a role for GalC or a highly related molecule in HIV entry into neural cells.     

Human CD4 binds immunoglobulins

T cell glycoprotein CD4 binds to class II major histocompatibility molecules and to the human immunodeficiency virus (HIV) envelope protein gp120. Recombinant CD4 (rCD4) bound to polyclonal immunoglobulin (Ig) and 39 of 50 (78%) human myeloma proteins. This binding depended on the Fab and not the Fc portion of Ig and was independent of the light chain. Soluble rCD4, HIV gp120, and sulfated dextrans inhibited the CD4-Ig interaction. With the use of a panel of synthetic peptides, the region critical for binding to Ig was localized to amino acids 21 to 38 of the first extracellular domain of CD4. CD4-bound antibody (Ab) complexed with antigen approximately 100 times better than Ab alone. This activity may contribute to the Ab-mediated enhancement of cellular HIV interaction that appears to depend on a trimolecular complex of HIV, antibodies to gp120, and CD4.     

Induction of CD4+ human cytolytic T cells specific for HIV-infected cells by a gp160 subunit vaccine

Cytolytic T lymphocyte (CTL) responses were evaluated in humans immunized with recombinant human immunodeficiency virus type 1 (HIV) envelope glycoprotein gp160. Some vaccinees had gp160-specific CTLs that were shown by cloning to be CD4+. Although induced by exogenous antigen, most gp160-specific CTL clones also recognized gp160 synthesized endogenously in target cells. These clones lysed autologous CD4+ T lymphoblasts infected with HIV. Of particular interest were certain vaccine-induced clones that lysed HIV-infected cells, recognized gp160 from diverse HIV isolates, and did not participate in "innocent bystander" killing of noninfected CD4+ T cells that had bound gp120.     

Prevention of HIV-1 infection and preservation of CD4 function by the binding of CPFs to gp120

Infection by human immunodeficiency virus type-1 (HIV-1) is initiated when its envelope protein, gp120, binds to its receptor, the cell surface glycoprotein CD4. Small molecules, termed N-carbomethoxycarbonyl-prolyl-phenylalanyl benzyl esters (CPFs), blocked this binding. CPFs interacted with gp120 and did not interfere with the binding of CD4 to class II major histocompatibility complex molecules. One CPF isomer, CPF(DD), preserved CD4-dependent T cell function while inhibiting HIV-1 infection of H9 tumor cells and human T cells. Although the production of viral proteins in infected T cells is unaltered by CPF(DD), this compound prevents the spread of infection in an in vitro model system.     

Synergism between HIV gp120 and gp120-specific antibody in blocking human T cell activation

The human immunodeficiency virus (HIV) binds to CD4-positive cells through interaction of its envelope glycoprotein (gp120) with the CD4 molecule. CD4 is a prominent immunoregulatory molecule, and chronic exposure to antibody against CD4 (anti-CD4) has been shown to cause immunodeficiency in mice. T cell-dependent in vitro immune responses can also be inhibited by anti-CD4. Experimental findings reported here indicate that CD4-bound gp120 attracts gp120-specific antibodies derived from the blood of HIV-seropositive individuals to form a trimolecular complex with itself and CD4. Thus targeted to CD4, the gp120-specific antibody functions as an antibody to CD4; it cross-links and modulates the CD4 molecules and suppresses the activation of T cells as measured by mobilization of intracellular calcium (Ca2i+). The synergism between gp120 and anti-gp120 in blocking T cell activation occurs at low concentrations of both components. Neither gp120 nor anti-gp120 inhibits T cell activation by itself in the concentrations tested.     

Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen

The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).     

The MHC-binding and gp120-binding functions of CD4 are separable

CD4 is a cell surface glycoprotein that is thought to interact with nonpolymorphic determinants of class II major histocompatibility (MHC) molecules. CD4 is also the receptor for the human immunodeficiency virus (HIV), binding with high affinity to the HIV-1 envelope glycoprotein, gp120. Homolog-scanning mutagenesis was used to identify CD4 regions that are important in class II MHC binding and to determine whether the gp120 and class II MHC binding sites of CD4 are related. Class II MHC binding was abolished by mutations in each of the first three immunoglobulin-like domains of CD4. The gp120 binding could be abolished without affecting class II MHC binding and vice versa, although at least one mutation examined reduced both functions significantly. These findings indicate that, while there may be overlap between the gp120 and class II MHC binding sites of CD4, these sites are distinct and can be separated. Thus it should be possible to design CD4 analogs that can block HIV infectivity but intrinsically lack the ability to affect the normal immune response by binding to class II MHC molecules.     

HTLV-III/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope

Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro. Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen. This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule. In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not. This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor. A segment of the HTLV-III/LAV envelope produced in E. coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome.     

Protein design of an HIV-1 entry inhibitor

Human immunodeficiency virus type-1 (HIV-1) membrane fusion is promoted by the formation of a trimer-of-hairpins structure that brings the amino- and carboxyl-terminal regions of the gp41 envelope glycoprotein ectodomain into close proximity. Peptides derived from the carboxyl-terminal region (called C-peptides) potently inhibit HIV-1 entry by binding to the gp41 amino-terminal region. To test the converse of this inhibitory strategy, we designed a small protein, denoted 5-Helix, that binds the C-peptide region of gp41. The 5-Helix protein displays potent (nanomolar) inhibitory activity against diverse HIV-1 variants and may serve as the basis for a new class of antiviral agents. The inhibitory activity of 5-Helix also suggests a strategy for generating an HIV-1 neutralizing antibody response that targets the carboxyl-terminal region of the gp41 ectodomain.     

Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV

Cells infected with the human immunodeficiency virus (HIV) show decreased expression of the 58-kilodalton T4 (CD4) antigen on their surface. In this study, the effect of HIV infection on the synthesis of T4 messenger RNA (mRNA) and protein products was evaluated in T-cell lines. Metabolically labeled lysates from the T4+ cell line Sup-T1 were immunoprecipitated with monoclonal antibodies to T4. Compared with uninfected cells, HIV-infected Sup-T1 cells showed decreased amounts of T4 that coprecipitated with both the 120-kilodalton viral envelope and the 150-kilodalton envelope precursor molecules. In four of five HIV-producing T-cell lines studied, the steady-state levels of T4 mRNA were also reduced. Thus, the decreased T4 antigen on HIV-infected cells is due to at least three factors: reduced steady-state levels of T4-specific mRNA, reduced amounts of immunoprecipitable T4 antigen, and the complexing of available T4 antigen with viral envelope gene products. The data suggested that the T4 protein produced after infection may be complexed with viral envelope gene products within infected cells. Retroviral envelope-receptor complexes may thus participate in a general mechanism by which receptors for retroviruses are down-modulated and alterations in cellular function develop after infection.     

Enhancement of SIV infection with soluble receptor molecules

The CD4 receptor on human T cells has been shown to play an integral part in the human immunodeficiency virus type 1 (HIV-1) infection process. Recombinant soluble human CD4 (rCD4) was tested for its ability to inhibit SIVagm, an HIV-like virus that naturally infects African green monkeys, in order to define T cell surface receptors critical for SIVagm infection. The rCD4 was found to enhance SIVagm infection of a human T cell line by as much as 18-fold, whereas HIV-1 infection was blocked by rCD4. Induction of syncytium formation and de novo protein synthesis were observed within the first 24 hours after SIVagm infection, whereas this process took 4 to 6 days in the absence of rCD4. This enhancing effect could be inhibited by monoclonal antibodies directed to rCD4. The enhancing effect could be abrogated with antibodies from naturally infected African green monkeys with inhibitory titers of from 1:2,000 to 1:10,000; these antibodies did not neutralize SIVagm infection in the absence of rCD4. Viral enhancement of SIVagm infection by rCD4 may result from the modulation of the viral membrane through gp120-CD4 binding, thus facilitating secondary events involved in viral fusion and penetration.     

HIV-infected cells are killed by rCD4-ricin A chain

The gp120 envelope glycoprotein of the human immunodeficiency virus (HIV), which is expressed on the surface of many HIV-infected cells, binds to the cell surface molecule CD4. Soluble derivatives of recombinant CD4 (rCD4) that bind gp120 with high affinity are attractive vehicles for targeting a cytotoxic reagent to HIV-infected cells. Soluble rCD4 was conjugated to the active subunit of the toxin ricin. This conjugate killed HIV-infected H9 cells but was 1/1000 as toxic to uninfected H9 cells (which do not express gp120) and was not toxic to Daudi cells (which express major histocompatibility class II antigens, the putative natural ligand for cell surface CD4). Specific killing of infected cells can be blocked by rgp120, rCD4, or a monoclonal antibody to the gp120 binding site on CD4.     

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.     

Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells

The first step in the infection of human T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is attachment to the target cell receptor, the CD4 antigen. This step may be vulnerable to attack by antibodies, chemicals, or small peptides. Dextran sulfate (molecular weight approximately 8000), which has been given to patients as an anticoagulant or antilipemic agent for more than two decades, was found to block the binding of virions to various target T lymphocytes, inhibit syncytia formation, and exert a potent inhibitory effect against HIV-1 in vitro at concentrations that may be clinically attainable in human beings. This drug also suppressed the replication of HIV-2 in vitro. These observations could have theoretical and clinical implications in the strategy to develop drugs against HIV types 1 and 2.