Catherine M. Finnegan

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.

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

ABSTRACT β-Defensins are small (3 to 5 kDa in size) secreted antimicrobial and antiviral proteins that are components of innate immunity. β-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 μg/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 β-defensins, especially human β-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 β-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 β-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.

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

ABSTRACT 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 HIVHXB2 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°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.

Antigenic Properties of the Human Immunodeficiency Virus Envelope during Cell-Cell Fusion

ABSTRACT Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIVHXB2 envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4°C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.

Sphingomyelinase Restricts the Lateral Diffusion of CD4 and Inhibits Human Immunodeficiency Virus Fusion

ABSTRACT 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.

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 Cell Biology of HIV-I Entry

The interaction of HIV-1 envelope glycoproteins with CD4 and coreceptors triggers a barrage of conformational changes in HIV-1 gp120 and gp41, which lead to the gp41 six-helix bundle formation that drives the membrane merger and eventual fusion. Although significant progress has been made in understanding HIV fusion, little is known about the cell biological processes that impact on HIV entry. Manipulating lipids has yielded important insights into the role that membrane of the host cell plays in regulating the HIV-1 entry process. In this talk I will describe how lipid manipulation affects HIV-1 entry by 1) altering the disposition and lateral mobility of HIV-1 receptors, 2) endosomal re-routing, and 3) cytoskeletal remodeling. Manipulation of lipid metabolism may therefore constitute a promising avenue for the development of antiretrovirals. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005