Antibodies against analogous heptad repeat peptide HR212 of Newcastle Disease Virus inhibit virus-cell membrane fusion

Membrane fusion is a key step in enveloped virus entry. Highly conserved heptad repeat regions (HR1 and HR2) of Newcastle disease virus (NDV) fusion protein (F) are critical functional domains for viral membrane fusion. They display different conformations in the membrane fusion states and are viewed as candidate targets for neutralizing antibody responses. We previously reported that an analog of heptad repeat peptides HR2-HR1-HR2(HR212) and HR2 could inhibit NDV induced cell-cell membrane fusion. Here, we show that HR212 can induce the production of highly potent antibody in immunized rabbits, which could recognize full length peptides of both HR1 and HR2, and inhibit NDV hemagglutination and NDV entry. These suggest that either HR212 or its antibody could be an inhibitor of virus-induced cell-cell membrane fusion.     

Interacting domains of the HN and F Proteins of paramyxovirus

Binding sialates to hemagglutinin-neuraminidase （HN） activates （triggers） the fusion protein （F） to start the membrane fusion process of paramyxovirus, but the mechanism by which the HN and F associate with each other to induce membrane fusion is still unclear. It is noteworthy to study the interaction domains of HN and F of paramyxovirus. To screen interacting domains of the HN and F proteins of Avian parainfluenza virus-2 （APIV-2） and identify the structure of binding proteins, the GST pull-down assay and mass spectroscopy （MS） and circular dichroism （CD） experiments were performed in this study. The study revealed that the globular head region of HN protein tends to form a complex with either the heptad repeat 1 （HR1） or the heptad repeat 2 （HR2） of F protein respectively. This paper discusses the novel fusion mechanism induced by paramyxovirus HN and F proteins.     

Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation

Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.     

A new mechanism for the neutralization of enveloped viruses by antiviral antibody

Despite the considerable research that has been carried out into viral neutralization by antiviral antibody, its mechanisms remain poorly understood. Cases have been reported in which antiviral antibody can inhibit viral replication without inhibiting the binding and uptake of virus by susceptible cells. It has been shown that many enveloped viruses enter their target cells by endocytosis and are subsequently located in cellular compartments of increasing acidity. With several enveloped viruses this acidic pH can catalyse a fusion reaction between the membrane of the virus particle and that of a prelysosomal endosome, thus enabling the viral core to enter the cytosol and replication to commence. We have recently demonstrated that such an endosomal fusion event at mild acidic pH is involved in the entry pathway of the enveloped flavivirus, West Nile virus (WNV), into macrophages. We now show that antiviral antibody can neutralize WNV by inhibiting this intraendosomal acid-catalysed fusion step and we speculate on possible implications for the future design of antiviral vaccines.     

A soluble CD4 protein selectively inhibits HIV replication and syncytium formation

The CD4 (T4) molecule is expressed on a subset of T lymphocytes involved in class II MHC recognition, and is probably the physiological receptor for one or more monomorphic regions of class II MHC (refs 1-3). CD4 also functions as a receptor for the human immunodeficiency virus (HIV) exterior envelope glycoprotein (gp120) (refs 4-9), being essential for virus entry into the host cell and for membrane fusion, which contributes to cell-to-cell transmission of the virus and to its cytopathic effects. We have used a baculovirus expression system to generate mg quantities of a hydrophilic extracellular segment of CD4. Concentrations of soluble CD4 in the nanomolar range, like certain anti-CD4 monoclonal antibodies, inhibit syncytium formation and HIV infection by binding gp120-expressing cells. Perhaps more importantly, class II specific T-cell interactions are uninhibited by soluble CD4 protein, whereas they are virtually abrogated by equivalent amounts of anti-T4 antibody. This may reflect substantial differences in CD4 affinity for gp120 and class II MHC.     

A peptide fusion protein inhibits angiogenesis and tumor growth by blocking VEGF binding to KDR

Vascular endothelial growth factor (VEGF) binding to its tyrosine kinase receptors (KDR/FLK1, Flt-1) induces angiogenesis. In search of the peptides blocking VEGF binding to its receptor KDR/FLK1 to inhibit tumorangiogenesis and growth, we screened a phage display peptide library with KDR as target protein, and some candidate peptides were isolated. In this study, we cloned the DNA fragment coding the peptide K237 from the library, into a vector pQE42 to express fusion protein DHFR-K237 in E. coli M15. The affection of fusion protein DHFR-K237 on endothelial cell proliferation and angiogenesis was investigated. In vitro, DHFR-K237 could completely block VEGF binding to KDR and significantly inhibit the VEGF-mediated proliferation of the human vascular endothelial cells. In vivo, DHFR-K237 inhibited angiogenesis in chick embryo chorioallantoric membrane and tumor growth in nude mice. These results suggest that K237 is an effective antagonist of VEGF binding to KDR, and could be a potential agent for cancer biotherapy.     

埃博拉病毒入侵宿主细胞的分子机制

 埃博拉病毒是一类能够感染并引起人和灵长类动物发生埃博拉出血热的烈性囊膜病毒。发现近40年中,埃博拉病毒给人类生命带来了极大威胁。然而,目前人们对于埃博拉病毒的了解非常有限,尤其是病毒与其宿主细胞受体的结合机制和膜融合机制相关信息的缺失,使得针对埃博拉病毒的特效药物的设计和研发工作阻碍重重。本文综述了埃博拉病毒分类、形态、病毒蛋白和病毒生命周期,着重介绍了高福院士团队在埃博拉病毒入侵宿主细胞的分子机制研究中的成果。通过结构学手段解析了埃博拉病毒激活态囊膜糖蛋白GPcl与宿主细胞受体NPC1分子的复合物结构,从原子水平上阐明了埃博拉病毒与宿主细胞相互识别的机制,并在结构基础上对病毒的膜融合促发机制做出推测,提出以埃博拉病毒为代表的新的（第5种）囊膜病毒膜融合激发机制,为抗埃博拉病毒药物和疫苗的设计提供了结构基础。     

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.     

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.     

Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor

Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unravelling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.     

Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells

Chromaffin cells release catecholamine- and peptide-containing granules by exocytosis, by a mechanism involving movement of secretory granules towards the cell membrane, their apposition to it and the fusion of the granule membrane with the plasma membrane. One of the two subunits of membrane-associated brain spectrin, alpha-fodrin is an actin-binding protein which is found at the periphery of chromaffin cells and may be involved in secretion. Because cultured chromaffin cells can be permeabilized with detergents, giving pores large enough to permit the entry of immunoglobulin molecules, we used permeabilized cells to test the effect of specific antibodies on secretory mechanisms. Incubation of permeabilized cells with polyclonal immunoaffinity-purified monospecific anti-alpha-fodrin antibody or its Fab fragments did not modify basal release but did specifically inhibit Ca2+-induced catecholamine release by exocytosis. Our observations indicate that fodrin and the cytoskeleton participate in the release mechanism.     

Evidence for existence of a close association between CD14 and CXCR4 on monocytic cell line U937

The surface expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocytes can be regulated by the ligand of CD14,and the susceptibility of the cells to HIV-1 is then changed.Our previous study found that monoclonal antibody against CD14 could dramatically inhibit CXCR4-mediated chemotaxis and cell-cell fusion.Based on these studies,we explored potential relationship between CD14 and CXCR4 on monocytic cell line U937.Flow cytometry analysis showed that anti-CXCR4 monoclonal antibody (mAb) 12G5 strongly inhibited binding of the FITC-conjugated anti-CD14 monoclonal antibodies (TUK4 and UCHM1) to U937,while another CX- CR4-specific mAb B-R24 did not show any effect on this binding.On the other hand,two anti-CD14 monoclonal antibodies (TUK4 and UCH-M1) obviously inhibited the binding of the PE-conjugated anti-CXCR4 mAb 12G5 to U937 but did not inhibit the binding of mAb 12G5 to CXCR4-transfected 3T3 cells (3T3.T4.CXCR4),which indicates that the blocking of mAb 12G5 binding to CXCR4 by CD14- specific mAbs is not involved in the possibility that CD14-specific mAbs directly bind to CXCR4.These results suggested existence of a close association between CD14 and CXCR4 on monocytic cell line U937.     

Evidence for existence of a close association between CD14 and CXCR4 on monocytic cell line U937

The surface expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocytes can be regulated by the ligand of CD14, and the susceptibility of the cells to HIV-1 is then changed. Our previous study found that monoclonal antibody against CD14 could dramatically inhibit CXCR4-mediated chemotaxis and cell-cell fusion. Based on these studies, we explored potential relationship between CD14 and CXCR4 on monocytic cell line U937. Flow cytometry analysis showed that anti-CXCR4 monoclonal antibody (mAb) 12G5 strongly inhibited binding of the FITC-conjugated anti-CD14 monoclonal antibodies (TUK4 and UCHM1) to U937, while another CXCR4-specific mAb B-R24 did not show any effect on this binding. On the other hand, two anti-CD14 monoclonal antibodies (TUK4 and UCH-M1) obviously inhibited the binding of the PE-conjugated anti-CXCR4 mAb 12G5 to U937 but did not inhibit the binding of mAb 12G5 to CXCR4-transfected 3T3 cells (3T3.T4.CXCR4), which indicates that the blocking of mAb 12G5 binding to CXCR4 by CD14-specific mAbs is not involved in the possibility that CD14-specific mAbs directly bind to CXCR4. These results suggested existence of a close association between CD14 and CXCR4 on monocytic cell line U937.     

Dissection of SARS Coronavirus Spike Protein into Discrete Folded Fragments

Introduction Severe acute respiratory syndrome coronavirus (SARS-CoV), which is the causative agent of the atypical pneumonia, was first identified in the fall of 2002 to be a previously unknown member of the family of coronaviruses[1]. The rapid transmis…     

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.     

Sperm antigen MSH27 participates in sperm-egg membrane fusion

A monoclonal antibody (mAb), MSH27 has been selected from an mAb library which has been prepared with separated mouse sperm heads as antigens. Three aspects of evidence indicated that the MSH27 antigen was involved in the process of sperm-egg membrane fusion. After the acrosome reaction, the antigen was located at sperm equatorial segment and in postacrosomal area, where, it was widely accepted, the sperm-egg membrane fusion initially occurred. In in vitro fertilization, the MSH27 antibody could decrease the index of sperm-egg membrane fusion, but made no effects on sperm approaching and binding to the plasma membrane of eggs. The inhibition showed an antibody concentration-dependent manner, with a rate decrease of 90% at 600 μg/mL immuno-globulin lgM. Furthermore, the antigen was able to affect the sperm-egg membrane fusion directly. The fusion index was obviously reduced after the zona-free eggs were exposed to the antigen purified by immuno-affinity chromatography. These, all together, demonstrated that the MSH27 antigen played an important role in sperm-egg membrane fusion.     

EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus

Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70 percent of infected patients, and there is evidence of human-to-human transmission. Endothelial syncytia, comprised of multinucleated giant-endothelial cells, are frequently found in NiV infections, and are mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the receptor for this virus will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. Here we report that ephrinB2, the membrane-bound ligand for the EphB class of receptor tyrosine kinases (RTKs), specifically binds to the attachment (G) glycoprotein of NiV. Soluble Fc-fusion proteins of ephrinB2, but not ephrinB1, effectively block NiV fusion and entry into permissive cell types. Moreover, transfection of ephrinB2 into non-permissive cells renders them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons, which is consistent with the known cellular tropism for NiV. Significantly, we find that NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons is inhibited by soluble ephrinB2, but not by the related ephrinB1 protein. Cumulatively, our data show that ephrinB2 is a functional receptor for NiV.     

Prevention of HIV-1 glycoprotein transport by soluble CD4 retained in the endoplasmic reticulum

The envelope glycoprotein (gp120/41) of the human immunodeficiency virus (HIV-1) attaches the virus to the cellular CD4 receptor and mediates virus entry into the cytoplasm. In addition to being required for formation of infectious HIV, expression of gp120/41 at the plasma membrane causes the cytopathic fusion of cells carrying the CD4 antigen. The expression of gp120/41 is therefore an ideal target for therapeutic strategies designed to combat AIDS. Here we show that expression of a soluble CD4 molecule, mutated to contain a specific retention signal for the endoplasmic reticulum, blocks secretion of gp120 and surface expression of gp120/41, but does not interfere with transport of wild-type CD4. By blocking transport of the HIV glycoprotein, this retained CD4 molecule prevents the fusion of CD4 cells that is normally caused by the HIV glycoprotein. Expression of the retained CD4 molecule in human T cells might therefore be useful in the intracellular immunization procedure suggested by Baltimore.     

Structural changes of envelope proteins during alphavirus fusion

Alphaviruses are enveloped RNA viruses that have a diameter of about 700?? and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.     

Structural rearrangements in the membrane penetration protein of a non-enveloped virus

Non-enveloped virus particles (those that lack a lipid-bilayer membrane) must breach the membrane of a target host cell to gain access to its cytoplasm. So far, the molecular mechanism of this membrane penetration step has resisted structural analysis. The spike protein VP4 is a principal component in the entry apparatus of rotavirus, a non-enveloped virus that causes gastroenteritis and kills 440,000 children each year. Trypsin cleavage of VP4 primes the virus for entry by triggering a rearrangement that rigidifies the VP4 spikes. We have determined the crystal structure, at 3.2 A resolution, of the main part of VP4 that projects from the virion. The crystal structure reveals a coiled-coil stabilized trimer. Comparison of this structure with the two-fold clustered VP4 spikes in a approximately 12 A resolution image reconstruction from electron cryomicroscopy of trypsin-primed virions shows that VP4 also undergoes a second rearrangement, in which the oligomer reorganizes and each subunit folds back on itself, translocating a potential membrane-interaction peptide from one end of the spike to the other. This rearrangement resembles the conformational transitions of membrane fusion proteins of enveloped viruses.