Sphingomyelinase restricts the lateral diffusion of CD4 and inhibits human immunodeficiency virus fusion.

Previously, we reported that treatment of cells with sphingomyelinase inhibits human immunodeficiency virus type 1 (HIV-1) entry. Here, we determined by measuring fluorescence recovery after photobleaching that the lateral diffusion of CD4 decreased 4-fold following sphingomyelinase treatment, while the effective diffusion rate of CCR5 remained unchanged. Notably, sphingomyelinase treatment of cells did not influence gp120 binding, HIV-1 attachment, or fluid-phase and receptor-mediated endocytosis. Furthermore, sphingomyelinase treatment did not affect the membrane disposition of the HIV receptor proteins CD4, CXCR4, and CCR5, as determined by Triton X-100 extraction. Restriction of CD4 diffusion by antibody cross-linking also inhibited HIV infection. We therefore interpret the decrease in CD4 lateral mobility following sphingomyelinase treatment in terms of clustering of CD4 molecules. Examination of fusion intermediates indicated that sphingomyelinase treatment inhibited HIV at a step in the fusion process after CD4 engagement. Maximal inhibition of fusion was observed following short coculture times and with target cells that express low levels of CD4. As HIV entry into cells requires the sequential engagement of viral envelope protein with CD4 and coreceptor, we propose that sphingomyelinase inhibits HIV infection by inducing CD4 clustering that prevents coreceptor engagement and HIV fusion.

Exposure of the membrane-proximal external region of HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion.

The membrane-proximal external region (MPER) of HIV-1 gp41 is highly conserved and critical for the fusogenic ability of the virus. However, little is known about the activity of this region in the context of viral fusion. In this study we investigate the temporal exposure of MPER during the course of HIV-1 Env-mediated fusion. We employed the broadly neutralizing monoclonal antibodies 2F5 and 4E10, whose epitopes localize to this region as indicators for accessibility to this region. Time of addition experiments indicated that escape of HIV-1 infection inhibition by 2F5 and 4E10 occurred concomitantly with that of C34, a peptide that blocks the six-helix bundle formation and fusion, which was about 20 min later than escape of inhibition by the mAb b12 that blocks CD4-gp120 attachment. We also probed accessibility of the MPER region on fusion intermediates by measuring the binding of the monoclonal antibodies at different time points during the fusion reaction. Immunofluorescence and in-cell Western assays showed that binding of 2F5 and 4E10 decreased upon triggering HIV-1 Env-expressing cells with appropriate target cells. Addition of C34 did not counteract the loss of antibody binding, suggesting that changes in exposure of MPER occur independently of six-helix bundle formation.

Fenretinide inhibits HIV infection by promoting viral endocytosis.

HIV fusion is mediated by the sequential interaction of the viral envelope glycoprotein with cellular receptors at the plasma membrane. We have previously reported that the upregulation of cellular ceramide levels following fenretinide treatment inhibits HIV fusion. As ceramide facilitates the internalization of a variety of microbes, we hypothesized that it may also promote the engulfment of HIV virions. Hence, we analyzed the effect of fenretinide treatment on virus binding and uptake. We observed that virus binding is not altered by fenretinide treatment. The distribution of HIV receptors was also unchanged. In contrast, virus uptake showed a significant increase. We have determined that fenretinide treatment promotes the internalization of virions from the plasma membrane and the accumulation of virus in the endocytic fraction of HeLa cells. This effect of fenretinide appears to be specific for virus as the endosomal accumulation of gp120, transferrin and horse-radish peroxidase was not increased. Notably, fenretinide increased the infectivity of influenza virus, which fuses in the endosomal compartment upon low pH activation. Our data suggest that fenretinide treatment effectively inhibits HIV infection by re-directing the virus to the endocytic pathway.

Human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection.

Beta-defensins are small (3 to 5 kDa in size) secreted antimicrobial and antiviral proteins that are components of innate immunity. Beta-defensins are secreted by epithelial cells, and they are expressed at high levels in several mucosae, including the mouth, where the concentration of these proteins can reach 100 microg/ml. Because of these properties, we wondered whether they could be part of the defenses that lower oral transmission of human immunodeficiency virus (HIV) compared to other mucosal sites. Our data show that select beta-defensins, especially human beta-defensin 2 (hBD2) and hBD3, inhibit R5 and X4 HIV infection in a dose-dependent manner at doses that are compatible with or below those measured in the oral cavity. We observed that beta-defensin treatment inhibited accumulation of early products of reverse transcription, as detected by PCR. We could not, however, detect any reproducible inhibition of env-mediated fusion, and we did not observe any modulation of HIV coreceptors following treatment with hBD1 and hBD2, in both resting and phytohemagglutinin-activated cells. Our data instead suggest that, besides a direct inactivation of HIV virions, hBD2 inhibits HIV replication in the intracellular environment. Therefore, we speculate that beta-defensins mediate a novel antiretroviral mechanism that contributes to prevention of oral HIV transmission in the oral cavity. Immunohistochemical data on hBD2 expression in oral mucosal tissue shows that hBD2 is constitutively expressed, forming a barrier layer across the epithelium in healthy subjects, while in HIV-positive subjects levels of hBD2 expression are dramatically diminished. This may predispose HIV-positive subjects to increased incidence of oral complications associated with HIV infection.

Ceramide, a target for antiretroviral therapy.

Studies of ceramide metabolism and function in a wide range of biological processes have revealed a role for this lipid in regulating key cellular responses. Our research on the role of sphingolipids in HIV entry has led to the hypothesis that modulation of ceramide levels in target cells affects their susceptibility to HIV infection by rearranging HIV receptors. Cellular ceramide levels were modulated by application of pharmacological agents such as N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide), by treatment with sphingomyelinase (Smase), or by exogenous addition of long-chain ceramide, and determined after metabolic incorporation of [3H]sphingosine. Infectivity assays were performed by using a HeLa-derived indicator cell line, TZM-bl, CD4+ lymphocytes, and monocytes. We observed a dose-dependent inhibition by 4-HPR of infection of TZM-bl cells by a broad range of HIV-1 isolates at low micromolar concentrations with an IC50 of <1 microM for most isolates tested. Nearly complete inhibition was seen at 5 microM, a dose that enhanced ceramide levels by 50-100%, yet was nontoxic to the cells. Treating cells with other pharmacological agents that enhanced ceramide levels, with Smase, or exogenous addition of long-chain ceramide also resulted in inhibition of HIV-1 infection. Enhancing ceramide levels in CD4+ lymphocytes and in monocyte-derived macrophages with 4-HPR or Smase significantly reduced infectivity without toxicity. The minimal toxicity of normal cells exposed to 4-HPR should make the drug exceedingly suitable as an anti-HIV therapeutic.

An inhibitor of glycosphingolipid metabolism blocks HIV-1 infection of primary T-cells.

OBJECTIVE: HIV-1 uses CD4 and chemokine receptors to enter cells. However, other target membrane components may also be involved. This study examines the role of glycosphingolipids (GSL) in HIV-1 entry into primary lymphocytes and its modulation by an inhibitor of GSL biosynthesis. METHODS: CD4 lymphocytes purified from normal or the p-group subtype individuals that were defective in Gb3 synthesis were treated with a GSL biosynthesis inhibitor, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP). The PPMP-treated cells were tested for HIV-1 replication by measuring p24 antigen production for 7-14 days post-infection and for susceptibility to HIV-1 Env-mediated fusion monitored by a fluorescent dye transfer assay. The effects of PPMP treatment on HIV-1 binding to CD4 lymphocytes were also examined by measuring HIV-1 p24. RESULTS: CD4 lymphocytes from p donors that are devoid of Gb3, but have elevated levels of GM3 were highly susceptible to HIV-1 fusion/entry. Pre-treatment of primary human CD4 lymphocytes from normal or p-sub-group type with PPMP, significantly reduced HIV-1 replication with no change in CD4 and CXCR4 levels. Inhibition of HIV-1 infection was due to the block in HIV-1 Env-mediated plasma membrane fusion. Binding of HIV-1 to CD4 lymphocytes was not affected by PPMP treatment. CONCLUSION: Manipulation of glycosphingolipid metabolic pathways may alter susceptibility of CD4 lymphocytes to HIV-1 entry.

The HIV Env-mediated fusion reaction.

The current general model of HIV viral entry involves the binding of the trimeric viral envelope glycoprotein gp120/gp41 to cell surface receptor CD4 and chemokine co-receptor CXCR4 or CCR5, which triggers conformational changes in the envelope proteins. Gp120 then dissociates from gp41, allowing for the fusion peptide to be inserted into the target membrane and the pre-hairpin configuration of the ectodomain to form. The C-terminal heptad repeat region and the leucine/isoleucine zipper region then form the thermostable six-helix coiled-coil, which drives the membrane merger and eventual fusion. This model needs updating, as there has been a wealth of data produced in the last few years concerning HIV entry, including target cell dependencies, fusion kinetic data, and conformational intermediates. A more complete model must include the involvement of membrane microdomains, actin polymerization, glycosphingolipids, and possibly CD4 and chemokine signaling in entry. In addition, kinetic experiments involving the addition of fusion inhibitors have revealed some of the rate-limiting steps in this process, adding a temporal component to the model. A review of these data that may require an updated version of the original model is presented here.

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion.

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.