Functional synergy between CD40 ligand and HIV-1 Tat contributes to inflammation: implications in HIV type 1 dementia

HIV type 1 (HIV-1)-associated dementia (HAD) is believed to occur due to aberrant activation of monocyte-derived macrophages and brain-resident microglial cells by viral proteins as well as by the proinflammatory mediators released by infected cells. To investigate the inflammatory aspects of the disease, we examined the levels of soluble CD40L (sCD40L) in paired samples of plasma and cerebrospinal fluid obtained from 25 HIV-infected individuals. A significantly higher level of sCD40L was detected in both cerebrospinal fluid and plasma from HIV-infected patients with cognitive impairment, compared with their nonimpaired counterparts. The contribution of sCD40L to the pathogenesis of HAD was then examined by in vitro experiments. rCD40L synergized with HIV-1 Tat to increase TNF-alpha release from primary human monocytes and microglia, in an NF-kappaB-dependent manner. The mechanistic basis for this synergism was attributed to a Tat-mediated up-regulation of CD40 in monocytes and microglia. Finally, the CD40L-mediated increase in TNF-alpha production by monocytes was shown to be biologically important; immunodepletion experiments revealed that TNF-alpha was essential for the neurotoxic effects of conditioned medium recovered from Tat/CD40L-treated monocytes. Taken together, our results show that CD40 signaling in microglia and monocytes can synergize with the effects of Tat, further amplifying inflammatory processes within the CNS and influencing neuronal survival.     

Interaction with damaged vessel wall in vivo in humans induces platelets to express CD40L resulting in endothelial activation with no effect of aspirin intake

Activated platelets express CD40L on their plasma membrane and release the soluble fragment sCD40L. The interaction between platelet surface CD40L and endothelial cell CD40 leads to the activation of endothelium contributing to atherothrombosis. Few studies have directly demonstrated an increased expression of platelet CD40L in conditions of in vivo platelet activation in humans, and no data are available on its relevance for endothelial activation. We aimed to assess whether platelets activated in vivo at a localized site of vascular injury in humans express CD40L and release sCD40L, whether the level of platelet CD40L expression attained in vivo is sufficient to induce endothelial activation, and whether platelet CD40L expression is inhibited by aspirin intake. We used the skin-bleeding-time test as a model to study the interaction between platelets and a damaged vessel wall by measuring CD40L in the blood emerging from a skin wound in vivo in healthy volunteers. In some experiments, shed blood was analyzed before and 1 h after the intake of 500 mg of aspirin. Platelets from the bleeding-time blood express CD40L and release soluble sCD40L, in a time-dependent way. In vivo platelet CD40L expression was mild but sufficient to induce VCAM-1 expression and IL-8 secretion in coincubation experiments with cultured human endothelial cells. Moreover, platelets recovered from the bleeding-time blood activated endothelial cells; an anti-CD40L antibody blocked this effect. On the contrary, the amount of sCD40L released by activated platelets at a localized site of vascular injury did not reach the concentrations required to induce endothelial cell activation. Soluble monocyte chemoattractant protein-1, a marker of endothelium activation, was increased in shed blood and correlated with platelet CD40L expression. Aspirin intake did not inhibit CD40L expression by platelets in vivo. We concluded that CD40L expressed by platelets in vivo in humans upon contact with a damaged vessel wall activates endothelium; aspirin treatment does not inhibit this mechanism.     

Involvement of nuclear factor κB in platelet CD40 signaling

CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-κB). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of IκBα, which are abolished by CD40L blockade. Inhibition of IκBα phosphorylation reverses sCD40L-induced IκBα phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of IκBα phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-κB activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo-inflammatory disorders.     

Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.     

Evidence that the species barrier of human immunodeficiency virus-1 does not extend to uptake by the blood--brain barrier: comparison of mouse and human brain microvessels

HIV-1 within the CNS produces a neuroAIDS syndrome and may act as a reservoir for reinfection of the peripheral tissues. Study of how HIV-1 crosses the blood-brain barrier (BBB) has been hampered by the lack of nonprimate animal models. However, BBB transport of HIV-1 does not involve any of the known steps conferring species specificity, including binding to CD4 receptors. In vivo and in vitro studies show that HIV-1 and its glycoprotein coat, gp120, are taken up and transported across the BBB of the mouse. Here, we compared the ability of gp120 and HIV-1 to be taken up by isolated brain microvessels (IBM) freshly isolated from mice, from post-mortem human brain, and from mice that had been treated in a manner analogous to the human material (mouse post-mortem). Freshly isolated mouse IBM took up more gp120 and HIV-1 than the human or mouse post-mortem cells. We found no difference between the ability of mouse post-mortem and human IBM to take up either gp120 or HIV-1. Wheatgerm agglutinin has been previously shown to stimulate gp120 and HIV-1 uptake by the BBB; here, it stimulated the uptake of gp120 and of HIV-1 by both mouse post-mortem and human IBM, although stimulated uptake was greatest for fresh mouse IBM. These results show that the mouse can be used to study the initial phases of HIV-1 uptake by the BBB.     

Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.     

Chronic immune activation in HIV-1 infection contributes to reduced interferon alpha production via enhanced CD40:CD40 ligand interaction

Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/µl, is not well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40, which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated HIV-1 infected individuals (n = 52) than in subjects on long-term antiretroviral therapy (n = 62, p<0.03) and in uninfected control donors (n = 16, p<0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC were significantly upregulated in HIV-1 infection (p<0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower, physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared to controls (p<0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p<0.001). In HIV-1 infected donors with less than 500 CD4+ T cells/µl, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-expressing PDC and the level of CD40 expression on these cells (p<0.05), whereas CD40L plasma levels played a minor role. In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.     

HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4(+) T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4(+) T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4(+) T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.     

Human immunodeficiency virus type 1 gp120 stimulates cytomegalovirus replication in monocytes: possible role of endogenous interleukin-8.

Recombinant gp120, but not other human immunodeficiency type 1 (HIV-1) structural proteins, dose-dependently stimulates human cytomegalovirus (HCMV) immediate-early antigen (IEA) expression and infectious virus yield in freshly isolated normal monocytes infected with HCMV. Monoclonal antibodies (MAbs) recognizing the gp120 V3 loop, as well as V3 loop octameric multibranched peptides and antibody to galactocerebroside, but not sCD4, abrogate the gp120 stimulation of IEA expression, suggesting that the effect involves V3 loop-galactocerebroside interaction and is not mediated by CD4. Interleukin 8 (IL-8) gene expression is enhanced in monocytes treated with gp120 at the level of both mRNA and released protein. Exogenous IL-8 could replace gp120 in the stimulation of HCMV infection, while a MAb capable of neutralizing IL-8 activity abrogates the gp120-induced HCMV stimulation. These data indicate that HIV-1 glycoprotein induces stimulation of productive infection of monocytes with HCMV and that such stimulation may be mediated by the upregulation of IL-8 gene expression. This is the first evidence that HIV-1 may affect HCMV replication indirectly, via the interaction of gp120 with the monocyte membrane, in the complete absence of retroviral replication, through the stimulation of IL-8 release. Because in HIV-1-infected individuals, HCMV infection is frequently activated and the levels of circulating IL-8 are enhanced, these findings may be pathogenetically relevant.     

Conformational changes induced in the envelope glycoproteins of the human and simian immunodeficiency viruses by soluble receptor binding.

We have investigated the molecular basis of biological differences observed among cell line-adapted isolates of the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and the simian immunodeficiency virus (SIV) in response to receptor binding by using a soluble form of CD4 (sCD4) as a receptor mimic. We find that sCD4 binds to the envelope glycoproteins of all of the HIV-1 isolates tested with affinities within a threefold range, whereas those of the HIV-2 and SIV isolates have relative affinities for sCD4 two- to eightfold lower than those of HIV-1. Treatment of infected cells with sCD4 induced the dissociation of gp120 from gp41 and increased the exposure of a cryptic gp41 epitope on all of the HIV-1 isolates. By contrast, neither dissociation of the outer envelope glycoprotein nor increased exposure of the transmembrane glycoprotein was observed when sCD4 bound to HIV-2- or SIV-infected cells. Moreover, immunoprecipitation with sCD4 resulted in the coprecipitation of the surface and transmembrane glycoproteins from virions of the HIV-2 and SIV isolates, whereas the surface envelope glycoprotein alone was precipitated from HIV-1. However, treatment of HIV-1-, HIV-2-, and SIV-infected cells with sCD4 did result in an increase in exposure of their V2 and V3 loops, as detected by enhanced antibody reactivity. This demonstrates that receptor binding to the outer envelope glycoprotein induces certain conformational changes which are common to all of these viruses and others which are restricted to cell line-passaged isolates of HIV-1.     

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.     

Innate and adaptive immune responses both contribute to pathological CD4 T cell activation in HIV-1 infected Ugandans

HIV-1 disease progression is associated with persistent immune activation. However, the nature of this association is incompletely understood. Here, we investigated immune activation in the CD4 T cell compartment of chronically HIV-1 infected individuals from Rakai, Uganda. Levels of CD4 T cell activation, assessed as co-expression of PD-1, CD38 and HLA-DR, correlated directly to viral load and inversely to CD4 count. Deeper characterization of these cells indicated an effector memory phenotype with relatively frequent expression of Ki67 despite their PD-1 expression, and levels of these cells were inversely associated with FoxP3+ regulatory T cells. We therefore use the term deregulated effector memory (DEM) cells to describe them. CD4 T cells with a DEM phenotype could be generated by antigen stimulation of recall responses in vitro. Responses against HIV-1 and CMV antigens were enriched among the DEM CD4 T cells in patients, and the diverse Vβ repertoire of DEM CD4 T cells suggested they include diverse antigen-specificities. Furthermore, the levels of DEM CD4 T cells correlated directly to soluble CD14 (sCD14) and IL-6, markers of innate immune activation, in plasma. The size of the activated DEM CD4 T cell subset was predictive of the rate of disease progression, whereas IL-6 was only weakly predictive and sCD14 was not predictive. Taken together, these results are consistent with a model where systemic innate immune activation and chronic antigen stimulation of adaptive T cell responses both play important roles in driving pathological CD4 T cell immune activation in HIV-1 disease.     

Glycoprotein 120 binding to CXCR4 causes p38-dependent primary T cell death that is facilitated by, but does not require cell-associated CD4

HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.     

Induction of SerpinB2 and Th1/Th2 Modulation by SerpinB2 during Lentiviral Infections In Vivo

SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic) SIV_mac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic) SHIV_mn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2^−/− mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2^−/− mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2^+/+ mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.     

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.     

A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4+ cells.

The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we show that, in contrast to the CD4 domain 1 MAb 6H10, 5A8 and its Fab fragment do not block soluble CD4 (sCD4) binding to virions, whereas they do inhibit sCD4-induced exposure of cryptic epitopes on gp41 and dissociation of gp120 from virions. Two other MAbs, OKT4 and L120, which are reactive with domains 3 and 4 of CD4, have little or no effect on HIV-1 infection, syncytium formation, or sCD4-induced conformational changes in the envelope glycoproteins. The mechanisms of action of 5A8 and 6H10 can be further distinguished in syncytium inhibition assays: 6H10 blocks competitively, while 5A8 does not. We opine that 5A8 blocks HIV-1 infection and fusion by interfering with conformational changes in gp120/gp41 and/or CD4 that are necessary for virus-cell fusion.