Variable constraints on the principal immunodominant domain of the transmembrane glycoprotein of human immunodeficiency virus type 1.
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Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex. (+info)
Interaction of peptides with sequences from the Newcastle disease virus fusion protein heptad repeat regions.
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Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix. (+info)
Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion.
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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. Munoz-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. (+info)
Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells.
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NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules. (+info)
Drainin required for membrane fusion of the contractile vacuole in Dictyostelium is the prototype of a protein family also represented in man.
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The contractile vacuole expels water by forming a channel with the plasma membrane and thus enables cells to survive in a hypo-osmotic environment. Here we characterize drainin, a Dictyostelium protein involved in this process, as the first member of a protein family represented in fission yeast, Caenorhabditis elegans and man. Gene replacement in Dictyostelium shows that drainin acts at a checkpoint of channel formation between the contractile vacuole and the plasma membrane. A green fluorescent protein fusion of drainin localizes specifically to the contractile vacuole and rescues its periodic discharge in drainin-null cells. Drainin is a peripheral membrane protein, requiring a short hydrophobic stretch in its C-terminal region for localization and function. We suggest that drainin acts in a signaling cascade that couples a volume-sensing device in the vacuolar membrane to the membrane fusion machinery. (+info)
Identification of Rab3A GTPase as an acrosome-associated small GTP-binding protein in rat sperm.
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The acrosome reaction is a membrane fusion event that is prerequisite for sperm penetration through the zona pellucida. To elucidate the molecular mechanisms involved in membrane fusion, the expression and localization of Rab proteins, a subfamily of small GTPases that have been shown to play key roles in regulation of intracellular membrane traffic and exocytosis, were examined in rat testis and sperm. Reverse transcription polymerase chain reaction, immunoblot analysis, and immunofluorescence microscopy revealed that Rab3A protein, which is thought to be involved in regulation of exocytosis in neurons and endocrine cells, is associated with the sperm acrosome. The protein was undetectable in acrosome-free heads prepared by sucrose density gradient centrifugation. Immunogold electron microscopy performed on ultrathin cryosections provided further evidence that Rab3A protein is associated with the acrosomal membrane. Acrosome reaction assays revealed that synthetic peptide of the Rab3 effector domain inhibited acrosomal exocytosis triggered by calcium ionophore A23187 in a concentration-dependent fashion, suggesting that Rab3A acts as an inhibitory regulator in the acrosome reaction. In view of the putative role of Rab3A protein in membrane fusion systems, these results suggest that Rab3A could be involved in regulating the mammalian acrosome reaction by controlling the membrane fusion system in sperm. (+info)
Molecular level investigation of exocytosis in cultured human pheochromocytoma cells: insights from high resolution scanning electron microscopy combined with autoradiogram and cytochemistry.
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OBJECTIVE: To investigate molecular events of exocytosis in cultured human pheochromocytoma cells with stimulation. METHODS: The cultured pheochromocytoma cells prepared from human adrenal pheochromocytoma tumor were stimulated for the release of catecholamines by depolarization with the administration of 50 mmol/L KCl. Transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HR-SEM) combined with autoradiography and cytochemistry were used to observe molecular mechanisms of exocytotic release of catecholamines from the stimulated cells labelled with 3H-noradrenaline and the filipin-treated cells. RESULTS: TEM and HR-SEM observations of the stimulated cells labelled with 3H-noradrenaline revealed that the initial exocytotic fusion pores even less than 10 nm in diameter in human pheochromocytoma cells can be clearly observed in a single lipid bilayer. Furthermore, HR-SEM examinations of the filipin-treated cells showed that the derangement of the particles of the filipin-sterol complexes (FSCs) in the fused membranes of granule and plasma membranes occurred as the exocytotic fusion pores opened. In addition, the aggreates of the FSCs particles were consistently demonstrated around the openings of the differently sized closing exocytotic pores. CONCLUSIONS: Based on our results, it is suggested that the rearrangement of the sterol molecules in the fused membranes of granule and plasma membranes plays an important role in the opening and closing mechanisms of exocytotic fusion pores. We hope that morphological data obtained in this study can provide some new insights into the understanding of molecular mechanisms of exocytosis, particularly the opening and closing of exocytotic fusion pores in relation to the distribution of the membrane sterols. (+info)
N- and C-terminal external domains of human herpesvirus-6 glycoprotein H affect a fusion-associated conformation mediated by glycoprotein L binding the N terminus.
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Human herpesvirus-6 (HHV-6), like other betaherpesviruses, shows cell fusion with wild-type strains, and this cellular spread is mediated by the glycoprotein gH/gL complex. Anti-fusion monoclonal antibodies (MAbs) specific for HHV-6 glycoprotein gH inhibit infection and prevent cellular spread by syncytia formation. Reactivity of these MAbs with gH deletion mutants suggests a conserved C-terminal fusion-associated domain. A conserved motif here has an N-glycosylation site and characteristics of a beta turn. Motif deletion abrogated MAb recognition while co-expression with glycoprotein gL restored this conformational epitope, indicating the importance of folding and not glycosylation at this site. Our previous studies showed gL binding to gH at an N-terminal domain specific for betaherpesviruses. To further examine the function of this N-terminal domain, a betaherpesvirus-specific motif was deleted. This mutant gH still bound gL, and was recognized by the anti-fusion MAbs; however, recognition was now primarily in the immature form and reduced during processing to the mature form. A model is discussed whereby gL binding gH at the N-terminal domain acts to draw together the C-terminal extracellular domain and this interaction affects a functional conformation during glycoprotein maturation. (+info)