Fusion Cascade Research Articles

θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

When a sperm meets an egg: Block to polyspermy

Embryonic development is initiated after the fertilizing spermatozoon enters the egg and triggers a series of events known as egg activation. Activation results in an increase in intracellular calcium concentration, cortical granule exocytosis (CGE), cell cycle resumption and recruitment of maternal mRNA. CGE is an evolutionary developed mechanism that causes modification of the zona pellucida to prevent penetration of additional spermatozoa, ensuring successful egg activation and embryo development. The egg CGE is a unique and convenient mammalian model for studying the different proteins participating at the membrane fusion cascade, which, unlike other secretory cells, occurs only once in the egg's lifespan. This article highlights a number of proteins, ascribed to participate in CGE and thus the block to polyspermy. CGE can be triggered either by a calcium dependent pathway, or via protein kinase C (PKC) activation that requires a very low calcium concentration. In a recent study, we suggested that the filamentous actin (F-actin) at the egg's cortex is a dynamic network. It can be maneuvered towards allowing CGE by activated actin associated proteins and/or by activated PKC and its down stream proteins, such as myristoylated alanine-rich C kinase substrate (MARCKS). MARCKS, a protein known to cross-link F-actin in other cell types, was found to be expressed and colocalized with actin in non-activated MII eggs. We further demonstrated MARCKS dissociation from actin after activation by ionomycin, a process that can lead to the breakdown of the actin network, thus allowing CGE. The more we know of the intricate process of CGE and of the proteins participating in it, the more the assisted reproductive procedures might benefit from that knowledge.

Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.

Dynamics of SNARE Assembly and Disassembly during Sperm Acrosomal Exocytosis

The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle—the acrosome—is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/α-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)–specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation.

Human immunodeficiency virus type 1 attachment, coreceptor, and fusion inhibitors are active against both direct and trans infection of primary cells.

Inhibitors of human immunodeficiency virus type 1 attachment (CD4-immunoglobulin G subclass 2), CCR5 usage (PRO 140), and fusion (T-20) were tested on diverse primary cell types that represent the major targets both for infection in vivo and for the inhibition of trans infection of target cells by virus bound to dendritic cells. Although minor cell-type-dependent differences in potency were observed, each inhibitor was active on each cell type and trans infection was similarly vulnerable to inhibition at each stage of the fusion cascade.

In vitro Fusion of Human Inguinal Hernia with Associated Epithelial Transformation

The processus vaginalis (PV) is a peritoneal diverticulum which forms to allow descent of the fetal testis to the scrotum. During human development fusion and obliteration of the PV often fails to occur with the result that inguinal hernias are the most prevalent congenital abnormality requiring surgery in childhood. Androgen is proposed to regulate testicular descent via the genitofemoral nerve which releases the neuropeptide calcitonin gene-related peptide (CGRP). It is possible that subsequent fusion of the PV and tissue remodelling following descent is indirectly controlled by androgen via CGRP action. An organ culture assay was developed to assess fusion of the PV taken from inguinal herniotomy in infants. Fusion was induced in vitro by CGRP but not by CGRP 8–37, CGRP 27–37 or dihydrotestosterone in equimolar concentrations. Fusion was accompanied by transformation of the epithelium, as shown by staining of intermediate filament proteins, cytokeratin and vimentin. Localization studies for CGRP receptors on 25 specimens indicated CGRP acts on mesenchymal fibroblasts but not directly on PV epithelium suggesting an indirect pathway. Hepatocyte growth factor/scatter factor was found to induce fusion of PV and may be involved as an intermediate molecule in the fusion cascade. This study represents the first approach to understanding the humoral control and underlying mechanism by which the PV fuses.

Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion.

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315-323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.

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.