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PubMed2003

Fusion peptides and the mechanism of viral fusion.

Segments of viral fusion proteins play an important role in viral fusion. They are defined by a number of criteria, including the sensitivity of this region of the viral fusion protein to loss of function as a consequence of mutation. In addition, small model peptides designed to mimic this segment of viral fusion proteins often have some membrane perturbing activity. The properties of viral fusion peptides are quite varied. Many are found at the amino terminus of viral fusion proteins. As isolated peptides, they have been found to form both alpha-helical as well as beta-structure. In addition, some viruses have internal fusion peptides. Just as there are several structural motifs for viral fusion peptides, there are also several mechanisms by which they accelerate the process of membrane fusion. These include the promotion of negative curvature, lowering the rupture tension of the lipid monolayer, acting as an anchor to join the fusion membranes, transmitting a force to the membrane or imparting energy to the system by other means. It is not likely that the fusion peptide can fulfill all of these diverse roles and future studies will elucidate which of these mechanisms is most important for the action of individual viral fusion peptides.

Lipid Bilayers↗

PubMed2001

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.

Hemagglutinin Glycoproteins, Influenza Virus↗

PubMed2002

Influence of gp41 fusion peptide on the kinetics of poly(ethylene glycol)-mediated model membrane fusion.

The fusion peptide of the HIV fusion protein gp41 is required for viral fusion and entry into a host cell, but it is unclear whether this 23-residue peptide can fuse model membranes. We address this question for model membrane vesicles in the presence and absence of aggregating concentrations of poly(ethylene glycol) (PEG). PEG had no effect on the physical properties of peptide bound to membranes or free in solution. We tested for fusion of both highly curved and uncurved PC/PE/SM/CH (35:30:15:20 mol %) vesicles and highly curved PC/PE/CH (1:1:1) vesicles treated with peptide in the presence and absence of PEG. Fusion was never observed in the absence of PEG, although high peptide concentrations led to aggregation and rupture, especially in unstable PC/PE/CH (1:1:1) vesicles. When 5 wt % PEG was present to aggregate vesicles, peptide enhanced the rate of lipid mixing between curved PC/PE/SM/CH vesicles in proportion to the peptide concentration, with this effect leveling off at peptide/lipid (P/L) ratios approximately 1:200. Peptide produced an even larger effect on the rate of contents mixing but inhibited contents mixing at P/L ratios >1:200. No fusion enhancement was seen with uncurved vesicles. The rate of fusion was also enhanced by the presence of hexadecane, and peptide-induced rate enhancement was not observed in the presence of hexadecane. We conclude that gp41 fusion peptide does not induce vesicle fusion at subrupturing concentrations but can enhance fusion between highly curved vesicles induced to fuse by PEG. The different effects of peptide on the rates of lipid mixing and fusion pore formation suggest that, while gp41 fusion peptide does affect hemifusion, it mainly affects pore formation.

Alkanes↗

PubMed2003

Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion.

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.

Amino Acid Sequence↗

PubMed1988

Separate epitopes in the envelope of visna virus are responsible for fusion and neutralization: biological implications for anti-fusion antibodies in limiting virus replication.

Visna virus is a lentivirus which causes fusion of infected cells in vitro. Two types of fusion occur. Fusion from without requires no viral replication and a relatively high multiplicity of infection; fusion from within results from the replication of virus in cells. By using fusion from without as an assay, the mechanism of fusion by visna virus was investigated. Immune sera which contained both anti-fusion and neutralizing antibodies interacted with the virus with rapid kinetics in blocking fusion but relatively slow kinetics in the virus neutralization assay. By using visna virus and an antigenic variant, the epitopes responsible for fusion and virus neutralization were shown to be different. Antigenic variation of visna virus resulted in alteration of the neutralization epitope and conservation of the fusion epitope. This suggested that there were two populations of antibodies and that the viral epitopes for fusion and neutralization were separate. These data suggest that visna virus is capable of infecting cells via two pathways: one via the fusion site and the other via the viral epitope which mediates neutralization.

Antigens, Viral↗

PubMed1986

Polylysine induces pH-dependent fusion of acidic phospholipid vesicles: a model for polycation-induced fusion.

Polylysine induced aggregation and fusion of negatively charged small unilamellar phosphatidylcholine vesicles containing at least 10% anionic lipid. Aggregation was followed by absorbance changes and fusion was assayed both by electron microscopy and by fluorescence energy transfer between lipid probes. A method for preparing asymmetric vesicles, where the fluorescent probes were present only in the inner monolayer of the vesicle membrane, was developed. These vesicles were used to distinguish the inner and outer monolayer when measuring lipid mixing between vesicles. Since polylysine induced lipid mixing of both monolayers equally, fusion of these vesicles did occur. The extent of fusion was dependent on the charge ratio between bound polylysine and phosphatidylserine (PS) in the outer monolayer and was optimal at a ratio of about 1:1. Excess polylysine inhibited fusion. At a given concentration of polypeptide, fusion increased as the pH was lowered toward 3 with an apparent pKa near 4. Since this value is close to the pKa of the PS-carboxyl groups and far from the pKa of the lysine epsilon-amino groups, the pH dependence observed for fusion resides in the lipids rather than in the peptide. Fusion was dependent on the available lysine and not the size or molarity of the polypeptide. The data indicate that there must be sufficient sites on the vesicles and sufficient polypeptide to achieve effective aggregation. For fusion to occur after aggregation, charges on the vesicles must be neutralized either by polypeptide-PS interaction or by protonation of the PS carboxyl groups. Optimal conditions for fusion occur when charge neutralization is possible without completely covering the vesicles with polypeptide. The results are consistent with the notion that the polypeptide is necessary for fusion because of requirements for crosslinking, but limits fusion by steric inhibition.

Cations, Divalent↗

PubMed2006

Surgical treatment for unstable low-grade isthmic spondylolisthesis in adults: a prospective controlled study of posterior instrumented fusion compared with combined anterior-posterior fusion.

BACKGROUND CONTEXT: The surgical treatment for low-grade isthmic spondylolisthesis in adults with intractable lumbar pain is usually spinal fusion. It has been postulated that anterior column reconstruction may be relatively advantageous in those patients with unstable slips. PURPOSE: To compare the early and medium term treatment efficacy of two common fusion techniques in isthmic spondylolisthesis. STUDY DESIGN/SETTING: Prospective controlled trial comparing single-level posterior-lateral instrumented fusion with combined anterior and posterior-lateral instrumented fusion in sequential matched cohorts of patients with radiographically unstable isthmic spondylolisthesis. OUTCOME MEASURES: Primary outcome measure of success was an Oswestry Disability Index (ODI)<or=20. Secondary outcome measures included patient determined minimum-acceptable outcome on four questionnaires: pain intensity (visual analog scale), ODI, medication intake, and work status. Radiographic outcome of fusion was determined by radiographic union and motion on flexion/extension X-rays. Risk ratios (RRs) and 95% confidence intervals (CIs) were calculated for primary outcome of success for combined fusion compared with posterior fusion. METHODS: The study was conducted over a 6-year period. The first cohort of 50 consecutive patients was treated with a single-level instrumented posterior-lateral fusion; the second sequential cohort was treated with an anterior interbody fusion and the same posterior operation. Observations were made at baseline, 6 months, 1 year, and 2 years after surgery. Final radiographic assessment was made at 2 years after surgery. RESULTS: Baseline demographic and clinical factors were well-matched in the two cohorts. At 2 years, 46 posterior-only fusion subjects and 47 combined fusion subjects completed the full follow-up regimen. Outcomes were better by all measures at 6 months and 12 months in the anterior-posterior cohort. Comparing the primary outcome measure (ODI outcome<or=20) in the posterior versus the combined groups, success was achieved at 6 months in 11 versus 30 (RR=2.67, 95% CI 1.53, 4.67; p=.0001); at 1 year, 20 versus 34 (RR=1.66, 95% CI 1.14, 2.42; p<.005); and at 2 years, 29 versus 36 subjects (RR=1.21, 95% CI 0.93, 1.59; p=.14). At 6 months, 13 posterior-only and 25 combined group subjects had returned to work (RR 1.88, 95% CI 1.10, 3.21; p=.01). More patients achieved their preoperatively determined minimum-acceptable outcome at each time point. There were three nonunions in the posterior-alone cohort and one in the combined group. Serious complications and reoperations were similar in both groups. CONCLUSION: Outcomes up to 2 years were superior by clinically important differences after a combined anterior-posterior operation compared with posterior-alone surgery for unstable spondylolisthesis; however, between-group differences attenuated appreciably after 6 months. The apparent clinical and occupational benefits of combined fusion should be considered along with possible increases in minor complications and procedure-related costs.

Adult↗

PubMed2002

Structural characterizations of fusion peptide analogs of influenza virus hemagglutinin. Implication of the necessity of a helix-hinge-helix motif in fusion activity.

Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.

Amino Acid Motifs↗

PubMed2002

Low fusion rate after L5-S1 laparoscopic anterior lumbar interbody fusion using twin stand-alone carbon fiber cages.

STUDY DESIGN: Prospective study of a cohort of patients who underwent L5-S1 laparoscopic anterior lumbar interbody fusion. OBJECTIVES: To assess the fusion rate and the clinical outcome more than 2 years after L5-S1 laparoscopic anterior lumbar interbody fusion using twin stand-alone carbon-fiber cages. SUMMARY OF BACKGROUND DATA: The first reports on laparoscopic anterior lumbar interbody fusion using stand-alone cages appeared in 1995. Since then several articles have reported contradictory data regarding fusion rate. There are no publications describing the fusion rate of stand-alone lumbar carbon-fiber cages. METHODS: The authors evaluated 12 patients (mean age 36.5 years) in whom endoscopic L5-S1 anterior lumbar interbody fusion was performed using twin stand-alone laparoscopic carbon-fiber cages. Clinical evaluation was carried out prospectively by the use of three self-evaluation scales. Radiologic evaluation was performed by an independent radiologist using dynamic flexion-extension films and CT scans at 6 and 12 months after surgery and subsequently every year until fusion was demonstrated. RESULTS: After a mean follow-up of 36.6 months (range 24-63 months) the clinical condition of the patients was significantly better than their preoperative status: visual analog scale (P < 0.01), Prolo score (P < 0.05), and Waddell Disability Index (P < 0.01). L5-S1 mobility did not exceed 5 degrees in any dynamic study. However, the overall CT scan fusion rate at 2 years of follow-up was 16.6%. Three years after surgery, CT demonstrated fusion in one of five patients. CONCLUSION: Two years after endoscopic L5-S1 anterior lumbar interbody fusion using twin stand-alone laparoscopic carbon-fiber cages, the fusion rate was unacceptably low. However, the clinical outcomes of these patients were significantly improved compared with their preoperative status.

Adult↗

PubMed2002

Circumferential lumbar spinal fusion with Brantigan cage versus posterolateral fusion with titanium Cotrel-Dubousset instrumentation: a prospective, randomized clinical study of 146 patients.

STUDY DESIGN: A prospective randomized clinical study with a 2-year follow-up period was conducted. OBJECTIVE: To analyze the effects of circumferential fusion using ALIF radiolucent carbon fiber cages and titanium posterior instrumentation on functional outcome, fusion rate, complications, and lumbar lordosis. SUMMARY OF BACKGROUND DATA: Circumferential fusion has become a common procedure in lumbar spine fusion, both as a primary and salvage procedure. However, the claimed advantages of ALIF plus PLF over conventional PLF lack scientific documentation. METHODS: From April 1996 through November 1999, a total of 148 patients with severe chronic low back pain were randomly selected for either posterolateral lumbar fusion with titanium CD-Horizon (posterolateral group) or circumferential fusion with a ALIF Brantigan cage plus posterior instrumentation. The Dallas Pain Questionnaire (DPQ), the Low Back Pain Rating Scale (LBPR), and a questionnaire concerning work status assessed their outcomes. RESULTS: Both groups showed highly significant improvement in all four categories of life quality (DPQ) as well as in the back pain and leg pain index (LBPR), as compared with preoperative status. There was a clear tendency toward better overall functional outcome for patients with the circumferential procedure ( < 0.08), and this patient group also showed significantly less leg pain at the 1-year follow-up evaluation ( < 0.03) and less peak back pain at 2 years ( < 0.04). Sagittal lordosis was restored and maintained in the circumferential group ( < 0.01). The circumferential fusion patients showed a higher posterolateral fusion rate (92%) than the posterolateral group (80%)( < 0.04). The repeat operation rate including implant removal was significantly lower in the circumferential group (7%) ( < 0.009) than in the posterolateral group (22%). CONCLUSIONS: Circumferential lumbar fusion restored lordosis, provided a higher union rate with significantly fewer repeat operations, showed a tendency toward better functional outcome, and resulted in less peak back pain and leg pain than instrumented posterolateral fusion. The clinical perspective of the current study implies a recommendation to favor circumferential fusion as a definitive surgical procedure in complex lumbar pathology involving major instability, flatback, and previous disc surgery in younger patients, as compared with posterolateral fusion with pedicle screws alone.

Adult↗

PubMed2005

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion.

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.

Amino Acid Sequence↗

PubMed2002

Comparative evaluation of microdiscectomy only, autograft fusion, polymethylmethacrylate interposition, and threaded titanium cage fusion for treatment of single-level cervical disc disease: a prospective randomized study in 125 patients.

OBJECT: The need for interbody fusion or stabilization after anterior cervical microdiscectomy is still debated. The objectives of this prospective randomized study were 1) to examine whether combined interbody fusion and stabilization is more beneficial than microdiscectomy only (MDO) and 2) if fusion is found to be more beneficial than MDO, to determine which is the best method of fusion by comparing the results achieved using autologous bone graft (ABG), polymethylmethacrylate (PMMA) interposition, and threaded titanium cage (TTC). METHODS: A total of 125 patients with a single-level cervical disc disease were included in this prospective study. All patients were randomized and assigned to one of the four following groups: Group 1 (33 patients), MDO; Group 2 (30 patients), microdiscectomy followed by ABG; Group 3 (26 patients), microdiscectomy followed by injection of PMMA; and Group 4 (36 patients), microdiscectomy followed by placement of a TTC. Clinical outcome according to Odom criteria was summarized as 1) excellent and good or 2) satisfactory and poor. One-year follow-up examination was performed in 123 patients. Patients in the TTC group experienced a significantly better outcome 6 months after surgery (92% excellent and good results) compared with those in the MDO and ABG groups (72.7 and 66.6% excellent and good results, respectively). Twelve months after surgery there was still a significant difference in outcomes between the TTC group (94.4% excellent and good results) and the MDO group (75.5% excellent and good results). Outcome in patients treated with PMMA was comparable with that in those treated with TCC after 6 (91.6%) and 12 months (87.5%), but no segmental fusion was achieved. Differences compared with MDO and ABG were, however, not significant, which may be related to the smaller number of patients in the PMMA group. CONCLUSIONS: Interbody cage-assisted fusion yields a significantly better short- and intermediate-term outcome than MDO in terms of return to work, radicular pain, Odom criteria, and earlier fusion. In addition, the advantages of interbody cages over ABG fusion included better results in terms of return to work, Odom criteria, and earlier fusion after 6 months. These results suggest that interbody cage-assisted fusion is a promising therapeutic option in patients with single-level disc disease. Polymethylmethacrylate seems to be a good alternative to interbody cage fusion but is hindered by the absence of immediate fusion.

Adult↗

PubMed2001

Role of the Glu residues of the influenza hemagglutinin fusion peptide in the pH dependence of fusion activity.

To elucidate the role of the fusion peptide in influenza hemagglutinin (HA)-mediated fusion, we compared pH-dependent conformational changes and fusion mediated by wild-type and a mutant HA in which Glu residues at positions 11 and 15 of the fusion peptide are substituted for valine. The pH dependence of conformational changes and kinetics of fusion with erythrocytes was the same for both forms of HA. The time for commitment and the temperature dependence of HA-mediated fusion were also the same. However, striking differences were observed between wild-type and mutant fusion peptides in their interactions with lipid membranes at neutral and acidic pH. Since elimination of the negatively charged residues allows the exposed fusion peptide to penetrate the bilayer at pH values closer to neutral, but does not affect conformational changes and fusion activity in intact HA, we conclude that conformational changes are tightly coupled to fusion peptide insertion in the overall HA-mediated fusion cascade.

3T3 Cells↗

PubMed2001

The fusion protein of Peste des petits ruminants virus mediates biological fusion in the absence of hemagglutinin-neuraminidase protein.

To study the process of membrane fusion in Morbilliviruses, the fusion (F) glycoproteins of Peste des petits ruminants virus (PPRV) and Rinderpest virus (RPV) were expressed transiently in mammalian cells. The recombinant F proteins were found to be localized at the surface of transfected cells. The fusion activity, as evident from cell fusion assays and lysis of chicken erythrocytes, documented that transiently expressed PPRV F glycoprotein induces cell fusion in the absence of homotypic hemagglutinin-neuraminidase (HN) attachment glycoprotein. The coexpression of homotypic HN increased the extent of fusion by twofold, while the efficiency of fusion was found to be substantially enhanced. In contrast, in RPV F-expressing cells, fusion was detected only when homotypic hemagglutinin (H) or heterotypic HN protein was coexpressed. This differs from the strict type-specific requirement for the attachment protein as in the fusion process of most of the paramyxoviruses. Further, we demonstrate by fluorescence transfer experiments that while PPRV F brings about both hemifusion and complete fusion on its own, RPV F induces only hemifusion while it brings about complete fusion in the presence of homotypic or heterotypic attachment protein.

Animals↗

PubMed2003

Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion.

Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.

Amino Acid Motifs↗

PubMed2004

Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core.

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.

Amino Acid Motifs↗

PubMed2003

Triggering of human parainfluenza virus 3 fusion protein (F) by the hemagglutinin-neuraminidase (HN) protein: an HN mutation diminishes the rate of F activation and fusion.

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

Animals↗

PubMed2000

Persistent fusion pores but transient fusion in alveolar type II cells.

The release of vesicle contents following exocytotic fusion is limited by various factors including the size of the fusion pore. Fusion pores are channel-like, narrow structures after formation and proceed through semi-stable states ('fusion pore flickering'), unless they fully expand (full fusion) or close again (transient fusion). Partial release of vesicle contents may occur during transient fusion, which was described to last between milliseconds and seconds, depending on the size of the vesicle. We studied fusion pores in a slow-secreting lung epithelial cell (type II cell) using fluorescence staining of vesicle contents (surfactant) and fluorescence recovery after photobleaching (FRAP). Surfactant is a lipidic material, which is secreted into the alveolar lumen to reduce the surface tension in the lung. We found release of surfactant to be a slow process, which can last for hours. Accordingly, fusion pores in these cells are stable structures, which appear to be a barrier for release. FRAP measurements suggest that transient fusions occasionally take place in these long-lasting fusion pores, resulting in partial release of surfactant into the extracellular space. These data suggest that postfusion mechanisms may regulate the amount of secreted surfactant.

Animals↗