A human immunodeficiency virus-transgenic mouse model for assessing interventions that block microbial-induced proviral expression.

A human immunodeficiency virus (HIV) type 1-transgenic mouse line (166) that previously showed up-regulated expression of viral proteins and infectious particles after infection with pathogenic agents was tested as a model for screening the in vitro and in vivo efficacy of inhibitors of HIV-1 immune activation. Two types of interventions were assessed: use of either the immunosuppressive drug prednisolone or an HIV-1 envelope-targeted toxin (sCD4-PE40). Both agents inhibited lipopolysaccharide-induced p24 expression by splenocytes in vitro and, when administered to transgenic mice, suppressed the induction of plasma p24, as well as the ex vivo production of p24 and infectious virus stimulated by in vivo infection with Mycobacterium avium. Moreover, HIV-1 mRNA levels in the spleen were greatly reduced in mice treated with either agent. Because HIV-1 expression cannot be induced in T lymphocytes from line 166 mice, this model may be of particular advantage for testing interventions that target virus production by non-T cell virus reservoirs.

Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: functional complementation of specific defects in gp120 and gp41.

The envelope glycoprotein (Env) of HIV-1 is displayed on the surface of the virion or infected cell as an oligomer of multiple gp120/gp41 complexes. We sought to unravel the relationships between this oligomeric structure and the requirements for sequential interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a quantitative cell fusion assay to examine the effects of coexpressing pairs of Envs, each nonfunctional because of a specific defect in one of the essential properties. We observed efficient fusion activity upon coexpression of two Env variants, one containing a gp41 subunit with a mutated fusion peptide and the other containing a gp120 subunit with a mutated CD4 binding site or a mismatched coreceptor specificity. We also observed fusion upon coexpression of two Env variants with distinct gp120 defects, i.e., a CD4 binding site mutation and the incorrect coreceptor specificity determinants. Coimmunoprecipitation experiments verified the efficient formation of mixed oligomers, suggesting that the observed fusion reflected subunit complementation within the oligomeric complex. These results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. The implications of these findings for Env function and virus neutralization are discussed.

Multiple antiviral activities of cyanovirin-N: blocking of human immunodeficiency virus type 1 gp120 interaction with CD4 and coreceptor and inhibition of diverse enveloped viruses.

Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.

Chimeric toxins targeted to the human immunodeficiency virus type 1 envelope glycoprotein augment the in vivo activity of combination antiretroviral therapy in thy/liv-SCID-Hu mice.

Highly active antiretroviral therapy (HAART), which combines multiple inhibitors of essential human immunodeficiency virus type 1 (HIV-1) enzymes, induces dramatic and sustained viral load reductions in many people infected with HIV-1. However, reservoirs of infected cells capable of producing replication-competent virus persist even after years of HAART, preventing elimination of infection. CD4-PE40 and 3B3(Fv)-PE38, chimeric toxins designed to target the HIV envelope (Env), represent a complementary class of agents that selectively kill productively infected cells. To investigate whether these Env-targeted toxins might serve as adjuncts to HAART for the elimination of infected cells, we tested their ability to augment HAART efficacy in vivo by using a thy/liv SCID-hu mouse model. CD4-PE40 and 3B3(Fv)-PE38 markedly enhanced the capacity of HAART to suppress acute HIV-1 infection and improved HAART-mediated viral load reduction in mice with established HIV-1 infection. These results represent the first demonstration of in vivo anti-HIV-1 efficacy for Env-targeted toxins and support their potential therapeutic utility in combination with HAART.

Sequential CD4-coreceptor interactions in human immunodeficiency virus type 1 Env function: soluble CD4 activates Env for coreceptor-dependent fusion and reveals blocking activities of antibodies against cryptic conserved epitopes on gp120.

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems.

Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease.

In addition to CD4, the human immunodeficiency virus (HIV) requires a coreceptor for entry into target cells. The chemokine receptors CXCR4 and CCR5, members of the G protein-coupled receptor superfamily, have been identified as the principal coreceptors for T cell line-tropic and macrophage-tropic HIV-1 isolates, respectively. The updated coreceptor repertoire includes numerous members, mostly chemokine receptors and related orphans. These discoveries provide a new framework for understanding critical features of the basic biology of HIV-1, including the selective tropism of individual viral variants for different CD4+ target cells and the membrane fusion mechanism governing virus entry. The coreceptors also provide molecular perspectives on central puzzles of HIV-1 disease, including the selective transmission of macrophage-tropic variants, the appearance of T cell line-tropic variants in many infected persons during progression to AIDS, and differing susceptibilities of individuals to infection and disease progression. Genetic findings have yielded major insights into the in vivo roles of individual coreceptors and their ligands; of particular importance is the discovery of an inactivating mutation in the CCR5 gene which, in homozygous form, confers strong resistance to HIV-1 infection. Beyond providing new perspectives on fundamental aspects of HIV-1 transmission and pathogenesis, the coreceptors suggest new avenues for developing novel therapeutic and preventative strategies to combat the AIDS epidemic.

Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gp120 or soluble CD4-induced conformational changes in gp120.

Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4(+) T cells and did not mediate "fusion from without" of CD4(+) target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.

CD46 is a cellular receptor for human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is the etiologic agent of exanthema subitum, causes opportunistic infections in immunocompromised patients, and has been implicated in multiple sclerosis and in the progression of AIDS. Here, we show that the two major HHV-6 subgroups (A and B) use human CD46 as a cellular receptor. Downregulation of surface CD46 was documented during the course of HHV-6 infection. Both acute infection and cell fusion mediated by HHV-6 were specifically inhibited by a monoclonal antibody to CD46; fusion was also blocked by soluble CD46. Nonhuman cells that were resistant to HHV-6 fusion and entry became susceptible upon expression of recombinant human CD46. The use of a ubiquitous immunoregulatory receptor opens novel perspectives for understanding the tropism and pathogenicity of HHV-6.

Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1.

Fractalkine is a multimodular human leukocyte chemoattractant protein and a member of the chemokine superfamily. Unlike other human chemokines, the chemokine domain of fractalkine has three amino acids between two conserved cysteines, referred to as the CX3C motif. Both plasma membrane-associated and shed forms of fractalkine have been identified. Here, we show that the recombinant 76-amino acid chemokine domain of fractalkine is a potent and highly specific chemotactic agonist at a human orphan receptor previously named V28 or alternatively CMKBRL1 (chemokine beta receptor-like 1), which was shown previously to be expressed in neutrophils, monocytes, T lymphocytes, and several solid organs, including brain. CMKBRL1/V28 also functioned with CD4 as a coreceptor for the envelope protein from a primary isolate of HIV-1 in a cell-cell fusion assay, and fusion was potently and specifically inhibited by fractalkine. Thus CMKBRL1/V28 is a specific receptor for fractalkine, and we propose to rename it CX3CR1 (CX3C chemokine receptor 1), according to an accepted nomenclature system.

Reconsidering targeted toxins to eliminate HIV infection: you gotta have HAART.

The success of highly active anti-retroviral therapy (HAART) has inspired new concepts for eliminating HIV from infected individuals. A major obstacle is the persistence of long-lived reservoirs of latently infected cells that might become activated at some time after cessation of therapy. We propose that, in the context of treatment strategies to deliberately activate and eliminate these reservoirs, hybrid toxins targeted to kill HIV-infected cells be reconsidered in combination with HAART. Such combinations might also prove valuable in protocols aimed at preventing mother-to-child transmission and establishment of infection immediately after exposure to HIV. We suggest experimental approaches in vitro and in animal models to test various issues related to safety and efficacy of this concept.

CC chemokine receptors 1 and 3 are differentially regulated by IL-5 during maturation of eosinophilic HL-60 cells.

CC chemokine receptors 1 and 3 (CCR1 and CCR3) are expressed by eosinophils; however, factors regulating their expression and function have not previously been defined. Here we analyze chemokine receptor expression and function during eosinophil differentiation, using the eosinophilic cell line HL-60 clone 15 as a model system. RNA for CCR1, -3, -4, and -5 was not detectable in the parental cells, and the cells did not specifically bind CC chemokines. Cells treated with butyric acid acquired eosinophil characteristics; expressed mRNA for CCR1 and CCR3, but not for CCR4 or CCR5; acquired specific binding sites for macrophage-inflammatory protein-1alpha and eotaxin (the selective ligands for CCR1 and CCR3, respectively); and exhibited specific calcium flux and chemotaxis responses to macrophage-inflammatory protein-1alpha, eotaxin, and other known CCR1 and CCR3 agonists. CCR3 was expressed later and at lower levels than CCR1 and could be further induced by IL-5, whereas IL-5 had little or no effect on CCR1 expression. Consistent with the HIV-1 coreceptor activity of CCR3, HL-60 clone 15 cells induced with butyric acid and IL-5 fused with HeLa cells expressing CCR3-tropic HIV-1 envelope glycoproteins, and fusion was blocked specifically by eotaxin or an anti-CCR3 mAb. These data suggest that CCR1 and CCR3 are markers of late eosinophil differentiation that are differentially regulated by IL-5 in this model.

Patterns of CCR5, CXCR4, and CCR3 usage by envelope glycoproteins from human immunodeficiency virus type 1 primary isolates.

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.

Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein.

BACKGROUND: 3B3 is a high-affinity anti-gp120 antibody that neutralizes a wide range of primary and laboratory isolates of HIV-1. The parental antibody was isolated from a combinatorial phage display library constructed from bone marrow RNA of an HIV-infected individual. We have generated a highly active immunotoxin using the 3B3 single-chain Fv (scFv) which can specifically kill lymphocytes infected by HIV-1. MATERIALS AND METHODS: We used recombinant DNA technology to clone the Fv fragment of 3B3 and produce a single-chain Fv (scFv). 3B3 scFv was then fused to a truncated version of Pseudomonas exotoxin A (PE38), giving rise to a recombinant immunotoxin 3B3(Fv)-PE38 that was expressed in E. coli and purified to near homogeneity. RESULTS: 3B3(Fv)-PE38 binds with the same affinity as the parental Fab antibody to the MN strain of gp120. The immunotoxin specifically kills a gp120-expressing transfected cell line and a chronically HIV-infected lymphocytic cell line. The immunotoxin is very stable at 37 degrees C, retaining 80% of its original activity after 24 hr. CONCLUSIONS: Potent immunotoxins such as 3B3(Fv)-PE38 could be utilized in combination with multidrug cocktails that limit viral replication to help reduce viral reservoirs in patients with AIDS.

CC chemokine receptor 5-mediated signaling and HIV-1 Co-receptor activity share common structural determinants. Critical residues in the third extracellular loop support HIV-1 fusion.

There is a close correspondence between the ability of RANTES and macrophage inflammatory proteins 1alpha and 1beta to activate CC chemokine receptor 5 (CCR5) and the ability to inhibit CCR5-dependent membrane fusion mediated by the envelope glycoprotein of human immunodeficiency virus (HIV), type 1. This finding suggests that some of the structural determinants for CC chemokine/CCR5 interactions and CCR5 HIV-1 fusion co-receptor activity may be shared. Recent studies using human CCR5/CCR2B chimeras have suggested that the determinants of CCR5 co-receptor activity are complex and may involve multiple extracellular receptor domains and that viral co-receptor activity is dissociable from ligand-dependent signaling responses. However, conclusive evidence demonstrating an important role for the second and third extracellular regions of human CCR5 is lacking. Furthermore, to determine whether the determinants for CCR5 co-receptor activity overlap with those required for agonist activity, studies that compare the chemokine specificity for inhibition of envelope-mediated cell fusion and the agonist profile of chimeric receptors are necessary. In the present report, using a series of CCR5/CCR2B chimeras we ascribe an important role for the second and third extracellular loop of CCR5 in supporting the co-receptor activity of CCR5. We also provide evidence that the intracytoplasmic tail of CCR5 does not play an important role in supporting HIV-1 entry. The hypothesis that the structural determinants for CC chemokine/CCR5 interactions and CCR5 HIV-1 fusion co-receptor activity may be shared was confirmed by two novel observations: first, the fusion activity supported by two hybrid receptors could be inhibited by both RANTES and monocyte chemoattractant protein-1, chemokines specific to CCR5 and CCR2B, respectively; and second, the chemokine specificity for inhibition of envelope-mediated cell fusion matched the agonist profile of these hybrid receptors. These data shed new light on the structural determinants involved in these distinct activities of CCR5 and may have important implications for the development of CCR5-targeted anti-viral compounds.