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Sample records for forest virus fusion

  1. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

    PubMed Central

    Wahlberg, J M; Bron, R; Wilschut, J; Garoff, H

    1992-01-01

    Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction. Images PMID:1433520

  2. Role of spike protein conformational changes in fusion of Semliki Forest virus.

    PubMed

    Justman, J; Klimjack, M R; Kielian, M

    1993-12-01

    The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes. PMID:8230478

  3. The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

    SciTech Connect

    Liao Maofu; Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2005-02-05

    The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.

  4. Pseudorevertants of a Semliki Forest Virus Fusion-Blocking Mutation Reveal a Critical Interchain Interaction in the Core Trimer▿

    PubMed Central

    Liu, Catherine Y.; Besanceney, Christen; Song, Yifan; Kielian, Margaret

    2010-01-01

    Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein. PMID:20826687

  5. Class II virus membrane fusion proteins

    SciTech Connect

    Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2006-01-05

    Enveloped animal viruses fuse their membrane with a host cell membrane, thus delivering the virus genetic material into the cytoplasm and initiating infection. This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction. This review describes recent advances in our understanding of the structure and function of the class II fusion proteins of the alphaviruses and flaviviruses. Inhibition of the fusion protein refolding reaction confirms its importance in fusion and suggests new antiviral strategies for these medically important viruses.

  6. Fusion of Enveloped Viruses in Endosomes.

    PubMed

    White, Judith M; Whittaker, Gary R

    2016-06-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. PMID:26935856

  7. Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

    PubMed Central

    Klupp, Barbara G.; Nixdorf, Ralf; Mettenleiter, Thomas C.

    2000-01-01

    A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine

  8. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    SciTech Connect

    Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

    2010-01-20

    The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

  9. Structural characterization of Mumps virus fusion protein core

    SciTech Connect

    Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng . E-mail: Qiubs@sun.im.ac.cn; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn; Tien, Po . E-mail: tienpo@sun.im.ac.cn

    2006-09-29

    The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.

  10. Reduction of Influenza Virus Envelope's Fusogenicity by Viral Fusion Inhibitors.

    PubMed

    Rowse, Michael; Qiu, Shihong; Tsao, Jun; Yamauchi, Yohei; Wang, Guoxin; Luo, Ming

    2016-01-01

    During cell entry of an enveloped virus, the viral membrane must be fused with the cellular membrane. The virus envelope has a unique structure consisting of viral proteins and a virus-specific lipid composition, whereas the host membrane has its own structure with host membrane proteins. Compound 136 was previously found to bind in close proximity to the viral envelope and inhibit influenza virus entry. We showed here that the 136-treated influenza virus still caused hemolysis. When liposomes were used as the target membrane for 136-treated viruses, aberrant fusion occurred; few liposomes fused per virion, and glycoproteins were not distributed evenly across fusion complexes. Additionally, large fusion aggregates did not form, and in some instances, neck-like structures were found. Based on previous results and hemolysis, fusion inhibition by 136 occurs post-scission but prior to lipid mixing. PMID:27622947

  11. A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

    PubMed Central

    Koehler, Jeffrey W.; Smith, Jeffrey M.; Ripoll, Daniel R.; Spik, Kristin W.; Taylor, Shannon L.; Badger, Catherine V.; Grant, Rebecca J.; Ogg, Monica M.; Wallqvist, Anders; Guttieri, Mary C.; Garry, Robert F.; Schmaljohn, Connie S.

    2013-01-01

    For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors. PMID:24069485

  12. Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

    PubMed

    Calder, Lesley J; Rosenthal, Peter B

    2016-09-01

    The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy. PMID:27501535

  13. Characterization of Potent Fusion Inhibitors of Influenza Virus

    PubMed Central

    Rowse, Michael; Qiu, Shihong; Tsao, Jun; Xian, Tongmei; Khawaja, Sarah; Yamauchi, Yohei; Yang, Zhen; Wang, Guoxin; Luo, Ming

    2015-01-01

    New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes. PMID:25803288

  14. Characterization of potent fusion inhibitors of influenza virus.

    PubMed

    Rowse, Michael; Qiu, Shihong; Tsao, Jun; Xian, Tongmei; Khawaja, Sarah; Yamauchi, Yohei; Yang, Zhen; Wang, Guoxin; Luo, Ming

    2015-01-01

    New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes. PMID:25803288

  15. Molecular mechanism of respiratory syncytial virus fusion inhibitors

    PubMed Central

    Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

    2016-01-01

    Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. PMID:26641933

  16. Molecular mechanism of respiratory syncytial virus fusion inhibitors.

    PubMed

    Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

    2016-02-01

    Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. PMID:26641933

  17. La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic

    SciTech Connect

    Soldan, Samantha S.; Hollidge, Bradley S.; Wagner, Valentina; Weber, Friedemann; Gonzalez-Scarano, Francisco

    2010-09-01

    La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066-1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066-1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative to LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.

  18. Reversible conformational changes and fusion activity of rabies virus glycoprotein.

    PubMed Central

    Gaudin, Y; Tuffereau, C; Segretain, D; Knossow, M; Flamand, A

    1991-01-01

    In an attempt to understand the implication of the rabies virus glycoprotein (G) in the first steps of the viral cycle, we studied the pH dependence of virus-induced fusion and hemagglutination, as well as modifications of the structure and properties of the viral glycoprotein following pH acidification. Our results suggest that the G protein adopts at least three distinct configurations, each associated with different properties. At neutral pH, G did not fuse membranes or hemagglutinate erythrocytes. It was insensitive to digestion with bromelain and trypsin. At pH 6.4, the glycoprotein became sensitive to proteases. Hemagglutination was at its maximum and then sharply decreased with the pH. No fusion was detected. Aggregation of virus was also observed. The third configuration, at below pH 6.1, was associated with the appearance of fusion. Some neutralizing monoclonal antibodies were able to differentiate these three configurations. Preincubation of the virus at below pH 6 inhibited fusion, but this inhibition, like the structural modifications of the glycoprotein, was reversible when G was reincubated at neutral pH. Images PMID:1870204

  19. Measles Virus Fusion Protein: Structure, Function and Inhibition.

    PubMed

    Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C

    2016-04-01

    Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

  20. Measles Virus Fusion Protein: Structure, Function and Inhibition

    PubMed Central

    Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C.

    2016-01-01

    Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

  1. Intracellular processing of the Newcastle disease virus fusion glycoprotein

    SciTech Connect

    Morrison, T.; Ward, L.J.; Semerjian, A.

    1985-03-01

    The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with (/sup 3/H) fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.

  2. Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion

    PubMed Central

    Sharma, Nishi R.; Mani, Prashant; Nandwani, Neha; Mishra, Rajakishore; Rana, Ajay; Sarkar, Debi P.

    2010-01-01

    Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy. PMID:20164223

  3. GS-5806 Inhibits Pre- to Postfusion Conformational Changes of the Respiratory Syncytial Virus Fusion Protein

    PubMed Central

    Xing, Weimei; Niedziela-Majka, Anita; Wong, Jinny S.; Hung, Magdeleine; Brendza, Katherine M.; Perron, Michel; Jordan, Robert; Sperandio, David; Liu, Xiaohong; Mackman, Richard; Sakowicz, Roman

    2015-01-01

    GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity. PMID:26324264

  4. GS-5806 inhibits pre- to postfusion conformational changes of the respiratory syncytial virus fusion protein.

    PubMed

    Samuel, Dharmaraj; Xing, Weimei; Niedziela-Majka, Anita; Wong, Jinny S; Hung, Magdeleine; Brendza, Katherine M; Perron, Michel; Jordan, Robert; Sperandio, David; Liu, Xiaohong; Mackman, Richard; Sakowicz, Roman

    2015-11-01

    GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity. PMID:26324264

  5. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

    PubMed Central

    Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z. Hong; Broder, Christopher C.; Aguilar, Hector C.; Nikolov, Dimitar B.

    2015-01-01

    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein. PMID:26646856

  6. Henipavirus mediated membrane fusion, virus entry and targeted therapeutics.

    PubMed

    Steffen, Deborah L; Xu, Kai; Nikolov, Dimitar B; Broder, Christopher C

    2012-02-01

    The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G) and class I fusion (F) envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins. PMID:22470837

  7. Henipavirus Mediated Membrane Fusion, Virus Entry and Targeted Therapeutics

    PubMed Central

    Steffen, Deborah L.; Xu, Kai; Nikolov, Dimitar B.; Broder, Christopher C.

    2012-01-01

    The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G) and class I fusion (F) envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins. PMID:22470837

  8. A neutron study of the feline leukaemia virus fusion peptide: Implications for biological fusion?

    NASA Astrophysics Data System (ADS)

    Davies, Sarah M. A.; Darkes, Malcolm J. M.; Bradshaw, Jeremy P.

    Neutron diffraction studies were performed on stacked phospholipid bilayers to determine the effects of the feline leukaemia virus (FeLV) fusion peptide on membrane structure. Bilayers were composed of dioleoylphosphatidylcholine with 50% (mol) dioleoylphosphatidylglycerol. Neutron scattering profiles with peptide present showed an increase in scattering density in the lipid-tails region, whilst scattering by the lipid headgroup region was decreased. This is interpreted as a lowering of the packing density of the lipid headgroups and an increase in the packing density of the lipid tails. Modelling studies and experimental evidence have suggested that fusion peptides catalyse fusion by increasing the negative curvature of the target membrane's outer monolayer. Our results presented here add support to this hypothesis for the fusion mechanism. The 2H 2O scattering profile was also slightly perturbed in the lipid headgroup region with 1% (mol)FeLV fusion peptide present. The FeLV peptide had no significant effect on the organisation of bilayers containing only dioleoylphosphatidylcholine.

  9. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

    PubMed Central

    Watterson, Daniel; Robinson, Jodie; Chappell, Keith J.; Butler, Mark S.; Edwards, David J.; Fry, Scott R.; Bermingham, Imogen M.; Cooper, Matthew A.; Young, Paul R.

    2016-01-01

    Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

  10. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

    PubMed

    Watterson, Daniel; Robinson, Jodie; Chappell, Keith J; Butler, Mark S; Edwards, David J; Fry, Scott R; Bermingham, Imogen M; Cooper, Matthew A; Young, Paul R

    2016-01-01

    Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

  11. Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Viruses with Liposomes Containing Lipid-Conjugated Heparin

    PubMed Central

    Smit, Jolanda M.; Waarts, Barry-Lee; Kimata, Koji; Klimstra, William B.; Bittman, Robert; Wilschut, Jan

    2002-01-01

    Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses. PMID:12239287

  12. The Forest, The Fly, and the Virus?

    NASA Technical Reports Server (NTRS)

    Tucker, Compton J.; Pinzon, Jorge E.; Wilson, James M.

    2003-01-01

    All known outbreaks of Ebola have been linked to tropical forests. We undertook a study of environmental conditions associated with Ebola hemorrhagic fever after preliminary reports strongly suggested that simultaneous outbreaks occurred, during two limited time periods in the 1970s and 1990s, immediately following sudden transitions between dry and wet seasons.

  13. Early Events in Chikungunya Virus Infection—From Virus Cell Binding to Membrane Fusion

    PubMed Central

    van Duijl-Richter, Mareike K. S.; Hoornweg, Tabitha E.; Rodenhuis-Zybert, Izabela A.; Smit, Jolanda M.

    2015-01-01

    Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research. PMID:26198242

  14. Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

    SciTech Connect

    Kelsey, D.R.; Flanagan, T.D.; Young, J.E.; Yeagle, P.L. )

    1991-06-01

    Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.

  15. An interaction site of the envelope proteins of Semliki Forest virus that is preserved after proteolytic activation

    SciTech Connect

    Zhang Xinyong; Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2005-07-05

    Semliki Forest virus (SFV) membrane fusion is mediated by the viral E1 protein at acidic pH and regulated by the dimeric interaction of E1 with the E2 membrane protein. During low pH-triggered fusion, the E2/E1 heterodimer dissociates, freeing E1 to drive membrane fusion. E2 is synthesized as a precursor, p62, which is processed to mature E2 by the cellular protease furin. Both the dissociation of the p62/E1 dimer and the fusion reaction of p62 virus have a more acidic pH threshold than that of the mature E2 virus. We have previously isolated SFV mutations that allow virus growth in furin-deficient cells. Here we have used such pci mutations to compare the interactions of the p62/E1 and E2/E1 dimers. Our data suggest that there is an important p62/E1 dimer interaction site identified by an E2 R250G mutation and that this interaction is maintained after processing to the mature E2 protein.

  16. Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles.

    PubMed

    Stegmann, T; Hoekstra, D; Scherphof, G; Wilschut, J

    1986-08-25

    We have investigated the pH-dependent fusion activity of influenza virus toward human erythrocyte ghosts, utilizing a recently developed fluorescence assay, which permits continuous monitoring of the fusion reaction. The rate of fusion is negligible at neutral pH but shows a sharp increase at pH values just below 5.5. This pH dependence profile closely corresponds to that of virus-induced hemolysis. Fusion is rapidly inactivated by a low-pH preincubation of the virus alone either at 37 or at 0 degrees C. The presence of ghosts during this low-pH preincubation, carried out at 0 degree C under which condition there is hardly any fusion, causes a significant protection of the viral fusion activity against inactivation. Fusion initiated at low pH can be arrested instantaneously by readjustment of the pH to neutral. The characteristics of fusion of influenza virus with ghosts deviate from those of fusion with cardiolipin liposomes (Stegmann, T., Hoekstra, D., Scherphof, G., and Wilschut, J. (1985) Biochemistry 24, 3107-3113). Fusion with ghosts is consistent with a requirement of the well-documented pH-dependent conformational change in the viral hemagglutinin, whereas fusion with cardiolipin liposomes does not exhibit a strict dependence on the conformational change. Rather, the negative surface charge on the liposomes plays a critical role, as zwitterionic liposomes containing gangliosides show fusion behavior similar to that of erythrocyte ghosts. PMID:3733744

  17. Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins

    PubMed Central

    Yu, Chia-Yi; Liang, Jian-Jong; Li, Jin-Kun; Lee, Yi-Ling; Chang, Bi-Lan; Su, Chan-I; Huang, Wei-Jheng; Lai, Michael M. C.; Lin, Yi-Ling

    2015-01-01

    Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I–like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection. PMID:26717518

  18. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-08-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. PMID:1830113

  19. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed Central

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-01-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. Images PMID:1830113

  20. Fusion of Hyperspectral and InSAR Based Satellite Data for Forest Biomass Modelling

    NASA Astrophysics Data System (ADS)

    Kattenborn, Teja; Maack, Joachim; Enble, Fabian; Fassnacht, Fabian; Emert, Jorg; Koch, Barbara

    2014-11-01

    During the last decades the availability of different spaceborne EO-Sensors increased notably. Hence, a combination of data from different EO sensors might feature a valuable procedure for assessments of complex variables such as forest ressources. The present work deals with the estimation of forest biomass based on a fusion of optical (hyperspectral) and geometric (InSAR) data. Reference data of hard- and softwood forest stands near Karlsruhe, Germany were sampled during the satellite data acquisition in summer 2013. Random forest models were applied using bootstrapped plot data as well as spatially clustered plots, whereas predictive accuracy was assessed within 5-fold cross validation. The best model achieved an average R² of 0.73 and RMSE of 29.4 t / ha. In comparison to studies using solely one sensor the present data fusion proved to be a more accurate approach for biomass estimates. Further research will include biomass assessments in mountainous areas and tropical forest.

  1. Structural Plasticity of the Semliki Forest Virus Glycome upon Interspecies Transmission

    PubMed Central

    2014-01-01

    Cross-species viral transmission subjects parent and progeny alphaviruses to differential post-translational processing of viral envelope glycoproteins. Alphavirus biogenesis has been extensively studied, and the Semliki Forest virus E1 and E2 glycoproteins have been shown to exhibit differing degrees of processing of N-linked glycans. However the composition of these glycans, including that arising from different host cells, has not been determined. Here we determined the chemical composition of the glycans from the prototypic alphavirus, Semliki Forest virus, propagated in both arthropod and rodent cell lines, by using ion-mobility mass spectrometry and collision-induced dissociation analysis. We observe that both the membrane-proximal E1 fusion glycoprotein and the protruding E2 attachment glycoprotein display heterogeneous glycosylation that contains N-linked glycans exhibiting both limited and extensive processing. However, E1 contained predominantly highly processed glycans dependent on the host cell, with rodent and mosquito-derived E1 exhibiting complex-type and paucimannose-type glycosylation, respectively. In contrast, the protruding E2 attachment glycoprotein primarily contained conserved under-processed oligomannose-type structures when produced in both rodent and mosquito cell lines. It is likely that glycan processing of E2 is structurally restricted by steric-hindrance imposed by local viral protein structure. This contrasts E1, which presents glycans characteristic of the host cell and is accessible to enzymes. We integrated our findings with previous cryo-electron microscopy and crystallographic analyses to produce a detailed model of the glycosylated mature virion surface. Taken together, these data reveal the degree to which virally encoded protein structure and cellular processing enzymes shape the virion glycome during interspecies transmission of Semliki Forest virus. PMID:24467287

  2. Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity.

    PubMed Central

    Fredericksen, B L; Whitt, M A

    1995-01-01

    We have introduced amino acid substitutions into two regions of the extracellular domain of the vesicular stomatitis virus (VSV) glycoprotein (G protein) and examined the effect of these mutations on protein transport, low-pH-induced stability of G protein oligomers, and membrane fusion activity. We suggested previously that the region between amino acids 118 and 139 may be important for the membrane fusion activity of G protein, on the basis of the characterization of a fusion-defective G protein mutant (M. A. Whitt, P. Zagouras, B. Crise, and J. K. Rose, J. Virol. 64:4907-4913, 1990). It has also been postulated by others that this region as well as the region between amino acids 181 and 212 may constitute putative internal fusion domains of VSV G protein. In this report, we show that three different amino acids substitutions between residues 118 and 139 (G-124-->E, P-127-->D, and A-133-->K) either altered or abolished low-pH-dependent membrane fusion activity. In contrast, substitutions between residues 192 and 212 resulted either in G proteins that had wild-type fusion activity or in mutant proteins in which the mutation prevented transport of G protein to the cell surface. Two of the substitutions between residues 118 and 139 (G-124-->E and P-127-->D) resulted in G proteins that were fusion defective at pH 5.7, although syncytia were observed after cells were treated with fusion buffer at pH 5.5, albeit at levels significantly less than that induced by wild-type G protein. Interestingly, when either G-124-->E or P-127-->D was incorporated into tsO45 virions, the resulting particles were not infectious, presumably because the viral envelope was not able to fuse with the proper intracellular membrane. These results support the hypothesis that the region between amino acids 118 and 139 is important for the membrane fusion activity of VSV G protein and may constitute an internal fusion domain. PMID:7853475

  3. Genetic studies of cell fusion induced by herpes simplex virus type 1.

    PubMed Central

    Read, G S; Person, S; Keller, P M

    1980-01-01

    Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infections (complementation test). In single infections, fusion began 4 to 6 h after infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild-type infected cells. Fusion was decreased in mixed infections with the mutants and wild-type virus, but the mutants displayed a codominant fusion phenotype. Fusion was not decreased in mixed infection with pairs of mutants, indicating that the mutants, with one possible exception, are members of the same complementation group. A linkage map was established for six of the mutants by analysis of recombination frequencies. PMID:6251259

  4. Regional Distribution of Forest Height and Biomass from Multisensor Data Fusion

    NASA Technical Reports Server (NTRS)

    Yu, Yifan; Saatchi, Sassan; Heath, Linda S.; LaPoint, Elizabeth; Myneni, Ranga; Knyazikhin, Yuri

    2010-01-01

    Elevation data acquired from radar interferometry at C-band from SRTM are used in data fusion techniques to estimate regional scale forest height and aboveground live biomass (AGLB) over the state of Maine. Two fusion techniques have been developed to perform post-processing and parameter estimations from four data sets: 1 arc sec National Elevation Data (NED), SRTM derived elevation (30 m), Landsat Enhanced Thematic Mapper (ETM) bands (30 m), derived vegetation index (VI) and NLCD2001 land cover map. The first fusion algorithm corrects for missing or erroneous NED data using an iterative interpolation approach and produces distribution of scattering phase centers from SRTM-NED in three dominant forest types of evergreen conifers, deciduous, and mixed stands. The second fusion technique integrates the USDA Forest Service, Forest Inventory and Analysis (FIA) ground-based plot data to develop an algorithm to transform the scattering phase centers into mean forest height and aboveground biomass. Height estimates over evergreen (R2 = 0.86, P < 0.001; RMSE = 1.1 m) and mixed forests (R2 = 0.93, P < 0.001, RMSE = 0.8 m) produced the best results. Estimates over deciduous forests were less accurate because of the winter acquisition of SRTM data and loss of scattering phase center from tree ]surface interaction. We used two methods to estimate AGLB; algorithms based on direct estimation from the scattering phase center produced higher precision (R2 = 0.79, RMSE = 25 Mg/ha) than those estimated from forest height (R2 = 0.25, RMSE = 66 Mg/ha). We discuss sources of uncertainty and implications of the results in the context of mapping regional and continental scale forest biomass distribution.

  5. Genetic studies of cell fusion induced by herpes simplex virus type 1

    SciTech Connect

    Read, G.S.; Person, S.; Keller, P.M.

    1980-07-01

    Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild type-infected cells.

  6. Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

    PubMed Central

    Schmid, Erik; Zurbriggen, Andreas; Gassen, Uta; Rima, Bert; ter Meulen, Volker; Schneider-Schaulies, Jürgen

    2000-01-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected. PMID:10906209

  7. Herpes B Virus Utilizes Human Nectin-1 but Not HVEM or PILRα for Cell-Cell Fusion and Virus Entry

    PubMed Central

    Fan, Qing; Amen, Melanie; Harden, Mallory; Severini, Alberto; Griffiths, Anthony

    2012-01-01

    To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRα did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections. PMID:22345445

  8. Fusion of imaging spectroscopy and airborne laser scanning data for characterization of forest ecosystems - A review

    NASA Astrophysics Data System (ADS)

    Torabzadeh, Hossein; Morsdorf, Felix; Schaepman, Michael E.

    2014-11-01

    Forest ecosystems play an important role in the global carbon cycle and it is largely unknown how this role might be altered by transients imposed by global change and deforestation. Remote sensing can provide information on ecosystem state and functioning and, among others, two remote sensing techniques, airborne laser scanning (ALS) and imaging spectroscopy (IS), have been used to characterize forest ecosystems, both independently and combined in fusion approaches. However, the fusion of these datasets should make the best use of the complementarity of both sensors and provide better and more robust vegetation products in forested ecosystems. Similar to other data fusion approaches, satisfying results depend on choosing appropriate fusion levels and methods. In this review paper, we summarize and classify relevant studies that focused on forest characterization using combined ALS and IS data, limited to the last decade. We classified the approaches by fusion level (data or product level) and by choice of methods (physical or empirical methods). Five different categories of products (landcover maps, aboveground biomass, biophysical parameters, gross/net primary productivity and biochemical parameters), have been found as the main aspects of forest ecosystems studied so far. A qualitative accuracy analysis of the products exposed that currently landcover maps are profiting the most from ALS and IS data fusion, while there is room for improvements in respect to the other products, such as biophysical parameters. Only few studies using physical approaches were found, but we expect the use of such approaches will increase with the growing availability of physically based radiative transfer models that can simulate both, ALS and IS data.

  9. Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

    PubMed

    Hoekstra, D; Klappe, K

    1986-04-01

    A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost. PMID:3005662

  10. The role of fusion activity of influenza A viruses in their biological properties.

    PubMed

    Jakubcová, L; Hollý, J; Varečková, E

    2016-06-01

    Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity. PMID:27265461

  11. Evaluation of landsat and MODIS data fusion products for analysis of dryland forest phenology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Current satellite sensors provide data of insufficient spatial and temporal resolutions to fully characterize the patchy phenology patterns of dryland forests. The spatial and temporal adaptive reflectance fusion model (STARFM) is an algorithm that fuses Landsat 30 m data with MODIS 500 m data to pr...

  12. Fusion of Erythrocytes by Sendai Virus Studied by Immuno-Freeze-Etching

    PubMed Central

    Bächi, Thomas; Aguet, Michel; Howe, Calderon

    1973-01-01

    Extensive fusion of human erythrocytes agglutinated by Sendai virus was observed after 30 s of incubation at 37 C. Electron microscopy of thin sections failed to reveal the presence of virions, viral fragments, or discrete viral antigens reactive with ferritin-labeled antibody at the sites of fusion. Immuno-freezeetching of membrane surfaces demonstrated the dispersal of viral envelope antigens from what appeared to be original sites of viral attachment. Virus-induced clustering of membrane glycoproteins was interpreted as resulting from interaction of viral antigens with membrane receptor proteins and forming the structural basis for fusion of membranes with one another. Images PMID:4351454

  13. A sensor fusion field experiment in forest ecosystem dynamics

    NASA Technical Reports Server (NTRS)

    Smith, James A.; Ranson, K. Jon; Williams, Darrel L.; Levine, Elissa R.; Goltz, Stewart M.

    1990-01-01

    The background of the Forest Ecosystem Dynamics field campaign is presented, a progress report on the analysis of the collected data and related modeling activities is provided, and plans for future experiments at different points in the phenological cycle are outlined. The ecological overview of the study site is presented, and attention is focused on forest stands, needles, and atmospheric measurements. Sensor deployment and thermal and microwave observations are discussed, along with two examples of the optical radiation measurements obtained during the experiment in support of radiative transfer modeling. Future activities pertaining to an archival system, synthetic aperture radar, carbon acquisition modeling, and upcoming field experiments are considered.

  14. Interferon action on parental Semliki forest virus ribonucleic acid.

    PubMed

    Friedman, R M; Fantes, K H; Levy, H B; Carter, W B

    1967-12-01

    Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA. PMID:5621488

  15. Structure of the Newcastle disease virus F protein in the post-fusion conformation

    SciTech Connect

    Swanson, Kurt; Wen, Xiaolin; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S.

    2010-11-17

    The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.

  16. IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion

    PubMed Central

    Chin, Christopher R.; Savidis, George; Brass, Abraham L.; Melikyan, Gregory B.

    2014-01-01

    Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. PMID:24699674

  17. Estimation by radiation inactivation of the size of functional units governing Sendai and influenza virus fusion

    SciTech Connect

    Bundo-Morita, K.; Gibson, S.; Lenard, J.

    1987-09-22

    The target sizes associated with fusion and hemolysis carried out by Sendai virus envelope glycoproteins were determined by radiation inactivation analysis. The target size for influenza virus mediated fusion with erythrocyte ghosts at pH 5.0 was also determined for comparison. Sendai-mediated fusion with erythrocyte ghosts at pH 7.0 was likewise inactivated exponentially with increasing radiation dose, yielding a target size of 60 +/- 6 kDa, a value consistent with the molecular weight of a single F-protein molecule. The inactivation curve for Sendai-mediated fusion with cardiolipin liposomes at pH 7.0, however, was more complex. Assuming a multiple target-single hit model, the target consisted of 2-3 units of ca. 60 kDa each. A similar target was seen if the liposome contained 10% gangliosides or if the reaction was measured at pH 5.0, suggesting that fusion occurred by the same mechanism at high and low pH. A target size of 261 +/- 48 kDa was found for Sendai-induced hemolysis, in contrast with influenza, which had a more complex target size for this activity. Sendai virus fusion thus occurs by different mechanisms depending upon the nature of the target membrane, since it is mediated by different functional units. Hemolysis is mediated by a functional unit different from that associated with erythrocyte ghost fusion or with cardiolipin liposome fusion.

  18. The heads of the measles virus attachment protein move to transmit the fusion-triggering signal

    PubMed Central

    Navaratnarajah, Chanakha K; Oezguen, Numan; Rupp, Levi; Kay, Leah; Leonard, Vincent HJ; Braun, Werner; Cattaneo, Roberto

    2010-01-01

    The measles virus entry system, constituted of attachment (hemagglutinin, H) and fusion proteins, operates based on a variety of natural and targeted receptors. However, the mechanism triggering fusion of the viral envelope with the plasma membrane is not understood. Here we tested a model considering that the two heads of an H-dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion-triggering signal. Indeed, stabilizing the H-dimer interface by additional inter-molecular disulfide bonds prevented membrane fusion, an effect reversed by a reducing agent. Moreover, a membrane-anchored designated receptor efficiently triggered fusion, provided it engaged the H-dimer at locations proximal to where the natural receptors bind, and distal to the H-dimer interface. We discuss how receptors may force H-heads to switch partners and transmit the fusion-triggering signal. PMID:21217701

  19. Determination of the minimal fusion peptide of bovine leukemia virus gp30

    SciTech Connect

    Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert . E-mail: brasseur.r@fsagx.ac.be; Charloteaux, Benoit

    2007-04-13

    In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.

  20. Role of ribosomes in Semliki Forest virus nucleocapsid uncoating.

    PubMed Central

    Singh, I; Helenius, A

    1992-01-01

    The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosome-bound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80% of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded. Images PMID:1433506

  1. Chikungunya virus fusion properties elucidated by single-particle and bulk approaches.

    PubMed

    van Duijl-Richter, Mareike K S; Blijleven, Jelle S; van Oijen, Antoine M; Smit, Jolanda M

    2015-08-01

    Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome-virus fusion studies, in which the fusion behaviour of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an individual particle can be visualised. The combination of these approaches allowed us to obtain detailed insight into the kinetics, lipid dependency and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within 2 s after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to deduce that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process. PMID:25872739

  2. Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling▿

    PubMed Central

    Aguilar, Hector C.; Matreyek, Kenneth A.; Choi, Daniel Y.; Filone, Claire Marie; Young, Sophia; Lee, Benhur

    2007-01-01

    The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ∼20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r2 = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms. PMID:17301148

  3. Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

    PubMed Central

    Konduru, Krishnamurthy; Bradfute, Steven B.; Jacques, Jerome; Manangeeswaran, Mohanraj; Nakamura, Siham; Morshed, Sufi; Wood, Steven C.; Bavari, Sina

    2011-01-01

    Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use. PMID:21329775

  4. Residues in the hendra virus fusion protein transmembrane domain are critical for endocytic recycling.

    PubMed

    Popa, Andreea; Carter, James R; Smith, Stacy E; Hellman, Lance; Fried, Michael G; Dutch, Rebecca Ellis

    2012-03-01

    Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking. PMID:22238299

  5. Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

    PubMed Central

    Shirogane, Yuta; Suzuki, Satoshi O.; Ikegame, Satoshi; Koga, Ritsuko

    2013-01-01

    Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein. PMID:23255801

  6. The Rigid Amphipathic Fusion Inhibitor dUY11 Acts through Photosensitization of Viruses

    PubMed Central

    Vigant, Frederic; Hollmann, Axel; Lee, Jihye; Santos, Nuno C.; Jung, Michael E.

    2014-01-01

    Rigid amphipathic fusion inhibitors (RAFIs) are lipophilic inverted-cone-shaped molecules thought to antagonize the membrane curvature transitions that occur during virus-cell fusion and are broad-spectrum antivirals against enveloped viruses (Broad-SAVE). Here, we show that RAFIs act like membrane-binding photosensitizers: their antiviral effect is dependent on light and the generation of singlet oxygen (1O2), similar to the mechanistic paradigm established for LJ001, a chemically unrelated class of Broad-SAVE. Photosensitization of viral membranes is a common mechanism that underlies these Broad-SAVE. PMID:24284320

  7. ESTIMATION OF TROPICAL FOREST STRUCTURE AND BIOMASS FROM FUSION OF RADAR AND LIDAR MEASUREMENTS (Invited)

    NASA Astrophysics Data System (ADS)

    Saatchi, S. S.; Dubayah, R.; Clark, D. B.; Chazdon, R.

    2009-12-01

    Radar and Lidar instruments are active remote sensing sensors with the potential of measuring forest vertical and horizontal structure and the aboveground biomass (AGB). In this paper, we present the analysis of radar and lidar data acquired over the La Selva Biological Station in Costa Rica. Radar polarimetry at L-band (25 cm wavelength), P-band (70 cm wavelength) and interferometry at C-band (6 cm wavelength) and VV polarization were acquired by the NASA/JPL airborne synthetic aperture radar (AIRSAR) system. Lidar images were provided by a large footprint airborne scanning Lidar known as the Laser Vegetation Imaging Sensor (LVIS). By including field measurements of structure and biomass over a variety of forest types, we examined: 1) sensitivity of radar and lidar measurements to forest structure and biomass, 2) accuracy of individual sensors for AGB estimation, and 3) synergism of radar imaging measurements with lidar imaging and sampling measurements for improving the estimation of 3-dimensional forest structure and AGB. The results showed that P-band radar combined with any interformteric measurement of forest height can capture approximately 85% of the variation of biomass in La Selva at spatial scales larger than 1 hectare. Similar analysis at L-band frequency captured only 70% of the variation. However, combination of lidar and radar measurements improved estimates of forest three-dimensional structure and biomass to above 90% for all forest types. We present a novel data fusion approach based on a Baysian estimation model with the capability of incorporating lidar samples and radar imagery. The model was used to simulate the potential of data fusion in future satellite mission scenarios as in BIOMASS (planned by ESA) at P-band and DESDynl (planned by NASA) at L-band. The estimation model was also able to quantify errors and uncertainties associated with the scale of measurements, spatial variability of forest structure, and differences in radar and lidar

  8. Inhibition of endosomal fusion activity of influenza virus by Rheum tanguticum (da-huang).

    PubMed

    Lin, Ta-Jen; Lin, Chwan-Fwu; Chiu, Cheng-Hsun; Lee, Ming-Chung; Horng, Jim-Tong

    2016-01-01

    Rhubarb (Rheum tanguticum; da-huang in Chinese medicine) is a herbal medicine that has been used widely for managing fever and removing toxicity. In this study, we investigated how rhubarb inhibits influenza virus during the early stage of the infectious cycle using different functional assays. A non-toxic ethanolic extract of rhubarb (Rex) inhibited several H1N1 subtypes of influenza A viruses in Madin-Darby canine kidney cells, including strains that are clinically resistant to oseltamivir. Time course analysis of Rex addition showed that viral entry was one of the steps that was inhibited by Rex. We also confirmed that Rex effectively inhibited viral attachment and penetration into the host cells. The inhibition of red blood cell haemolysis and cell-cell fusion by Rex suggests that Rex may block haemagglutinin-mediated fusion (virus-endosome fusion) during the fusion/uncoating step. Rex has the capacity to inhibit influenza viruses by blocking viral endocytosis. Thus, rhubarb might provide an alternative therapeutic approach when resistant viruses become more prevalent. PMID:27302738

  9. Identification of Novel Fusion Inhibitors of Influenza A Virus by Chemical Genetics

    PubMed Central

    Lai, Kin Kui; Cheung, Nam Nam; Yang, Fang; Dai, Jun; Liu, Li; Chen, Zhiwei; Sze, Kong Hung; Chen, Honglin

    2015-01-01

    ABSTRACT A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses. IMPORTANCE Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets situated in close proximity to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests a close proximity and even partial overlap of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structure analyses of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of

  10. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    SciTech Connect

    Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  11. Role of the Amphipathic Peptide of Semliki Forest Virus Replicase Protein nsP1 in Membrane Association and Virus Replication▿

    PubMed Central

    Spuul, Pirjo; Salonen, Anne; Merits, Andres; Jokitalo, Eija; Kääriäinen, Leevi; Ahola, Tero

    2007-01-01

    Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein. PMID:17093195

  12. Nonreplicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins.

    PubMed

    Taylor, J; Weinberg, R; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1992-03-01

    The development of canarypox virus (CPV) recombinants expressing the hemagglutinin (HA) and fusion (F) glycoproteins of measles virus (MV) is described. Inoculation of the CPV-MV recombinants into avian or nonavian tissue culture substrates led to the expression of authentic MVF and MVHA as determined by radioimmunoprecipitation and surface immunofluorescence. In contrast to avian-derived tissue culture, no productive replication of the CPV recombinant was evident in tissue culture cells derived from nonavian origin. On inoculation of dogs, a species restricted for avipoxvirus replication, the recombinants elicited a protective immune response against a lethal canine distemper virus (CDV) challenge. The level of MV neutralizing antibodies and the level of protection induced against CDV challenge achieved by the host-restricted CPV vector were equivalent to that obtained by vaccinia virus vectors expressing the same MV antigens. PMID:1736535

  13. Inhibition of endosomal fusion activity of influenza virus by Rheum tanguticum (da-huang)

    PubMed Central

    Lin, Ta-Jen; Lin, Chwan-Fwu; Chiu, Cheng-Hsun; Lee, Ming-Chung; Horng, Jim-Tong

    2016-01-01

    Rhubarb (Rheum tanguticum; da-huang in Chinese medicine) is a herbal medicine that has been used widely for managing fever and removing toxicity. In this study, we investigated how rhubarb inhibits influenza virus during the early stage of the infectious cycle using different functional assays. A non-toxic ethanolic extract of rhubarb (Rex) inhibited several H1N1 subtypes of influenza A viruses in Madin–Darby canine kidney cells, including strains that are clinically resistant to oseltamivir. Time course analysis of Rex addition showed that viral entry was one of the steps that was inhibited by Rex. We also confirmed that Rex effectively inhibited viral attachment and penetration into the host cells. The inhibition of red blood cell haemolysis and cell–cell fusion by Rex suggests that Rex may block haemagglutinin-mediated fusion (virus–endosome fusion) during the fusion/uncoating step. Rex has the capacity to inhibit influenza viruses by blocking viral endocytosis. Thus, rhubarb might provide an alternative therapeutic approach when resistant viruses become more prevalent. PMID:27302738

  14. Newcastle disease virus fusion and haemagglutinin-neuraminidase proteins contribute to its macrophage host range

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic damage to international poultry industry. Different strains of NDV express a wide range of virulence that is primarily dependent on the amino acid sequence of the strain’s fusion (F) protein cleavage site. Two c...

  15. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    PubMed Central

    Gannagé, Monique; Schmid, Dorothee; Albrecht, Randy; Dengjel, Jörn; Torossi, Tania; Rämer, Patrick C.; Lee, Monica; Strowig, Till; Arrey, Frida; Conenello, Gina; Pypaert, Marc; Andersen, Jens; García-Sastre, Adolfo; Münz, Christian

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here we demonstrate that live influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes. Matrix protein 2 is sufficient and necessary for this inhibition of autophagosome degradation. Macroautophagy inhibition compromises cell survival of influenza virus infected cells, but does not influence viral replication. We propose that influenza A virus, which also encodes pro-apoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and blocking macroautophagy. PMID:19837376

  16. New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

    PubMed Central

    Antanasijevic, Aleksandar; Wang, Minxiu; Li, Bing; Mills, Debra M.; Ames, Jessica A.; Nash, Peter J.; Williams, John D.; Peet, Norton P.; Moir, Donald T.; Prichard, Mark N.; Keith, Kathy A.; Barnard, Dale L.; Caffrey, Michael; Rong, Lijun; Bowlin, Terry L.

    2014-01-01

    Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC50] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM2 % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process. PMID:24198411

  17. Nef Does Not Affect the Efficiency of Human Immunodeficiency Virus Type 1 Fusion with Target Cells

    PubMed Central

    Tobiume, Minoru; Lineberger, Janet E.; Lundquist, Christopher A.; Miller, Michael D.; Aiken, Christopher

    2003-01-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by an unknown mechanism. Recent studies have suggested that Nef may act by regulating the efficiency of virus entry into cells. Here we provide evidence to the contrary. Using a quantitative assay of HIV-1 virus-cell fusion, we observed equivalent rates and extents of fusion of wild-type and Nef-defective HIV-1 particles with MT-4 cells and CD4-expressing HeLa cells. In studies using soluble CD4 (sCD4) to inhibit infection, wild-type and Nef-defective HIV-1 escaped the sCD4 block with similar kinetics. We conclude that Nef acts at a postentry step in infection, probably by facilitating intracellular transport of the HIV-1 ribonucleoprotein complex. PMID:12970449

  18. Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

    PubMed Central

    Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.

    2016-01-01

    ABSTRACT Ebola virus (EBOV) makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP) to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveries, late events in EBOV entry following GP-NPC1 binding and culminating in GP-catalyzed fusion between viral and cellular lipid bilayers remain enigmatic. A mechanistic understanding of EBOV membrane fusion has been hampered by the failure of previous efforts to reconstitute fusion in vitro or at the cell surface. This report describes an assay to monitor initial steps directly in EBOV membrane fusion—triggering of GP and virus-cell lipid mixing—by single virions in live cells. Fusogenic triggering of GP occurs predominantly in Rab7-positive (Rab7+) endosomes, absolutely requires interaction between proteolytically primed GP and NPC1, and is blocked by key GP-specific neutralizing antibodies with therapeutic potential. Unexpectedly, cysteine protease inhibitors do not inhibit lipid mixing by virions bearing precleaved GP, even though they completely block cytoplasmic entry by these viruses, as shown previously. These results point to distinct cellular requirements for different steps in EBOV membrane fusion and suggest a model in which host cysteine proteases are dispensable for GP fusion triggering after NPC1 binding but are required for the formation of fusion pores that permit genome delivery. PMID:26861015

  19. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    PubMed Central

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  20. Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

    SciTech Connect

    Corey, Elizabeth A.; Iorio, Ronald M.

    2009-01-05

    Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation of fusion by these two paramyxoviruses.

  1. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

    PubMed Central

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

    2015-01-01

    ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1

  2. A Novel Influenza Virus Hemagglutinin-Respiratory Syncytial Virus (RSV) Fusion Protein Subunit Vaccine against Influenza and RSV

    PubMed Central

    Turner, Tiffany M.; Jones, Les P.; Tompkins, S. Mark

    2013-01-01

    Influenza A virus and respiratory syncytial virus (RSV) cause substantial morbidity and mortality afflicting the ends of the age spectrum during the autumn through winter months in the United States. The benefit of vaccination against RSV and influenza using a subunit vaccine to enhance immunity and neutralizing antibody was investigated. Influenza virus hemagglutinin (HA) and RSV fusion (F) protein were tested as vaccine components alone and in combination to explore the adjuvant properties of RSV F protein on HA immunity. Mice vaccinated with HA and F exhibited robust immunity that, when challenged, had reduced viral burden for both influenza and RSV. These studies show an enhancing and cross-protective benefit of F protein for anti-HA immunity. PMID:23903841

  3. Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

    PubMed Central

    Costin, Joshua M.; Zaitseva, Elena; Kahle, Kristen M.; Nicholson, Cindo O.; Rowe, Dawne K.; Graham, Amanda S.; Bazzone, Lindsey E.; Hogancamp, Greg; Figueroa Sierra, Marielys; Fong, Rachel H.; Yang, Sung-Tae; Lin, Li; Robinson, James E.; Doranz, Benjamin J.; Chernomordik, Leonid V.; Michael, Scott F.; Schieffelin, John S.

    2013-01-01

    There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies. PMID:23077306

  4. Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

    PubMed Central

    2012-01-01

    Background Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. Methods A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity. Results Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178

  5. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    SciTech Connect

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  6. Sequential Monte Carlo tracking of the marginal artery by multiple cue fusion and random forest regression.

    PubMed

    Cherry, Kevin M; Peplinski, Brandon; Kim, Lauren; Wang, Shijun; Lu, Le; Zhang, Weidong; Liu, Jianfei; Wei, Zhuoshi; Summers, Ronald M

    2015-01-01

    Given the potential importance of marginal artery localization in automated registration in computed tomography colonography (CTC), we have devised a semi-automated method of marginal vessel detection employing sequential Monte Carlo tracking (also known as particle filtering tracking) by multiple cue fusion based on intensity, vesselness, organ detection, and minimum spanning tree information for poorly enhanced vessel segments. We then employed a random forest algorithm for intelligent cue fusion and decision making which achieved high sensitivity and robustness. After applying a vessel pruning procedure to the tracking results, we achieved statistically significantly improved precision compared to a baseline Hessian detection method (2.7% versus 75.2%, p<0.001). This method also showed statistically significantly improved recall rate compared to a 2-cue baseline method using fewer vessel cues (30.7% versus 67.7%, p<0.001). These results demonstrate that marginal artery localization on CTC is feasible by combining a discriminative classifier (i.e., random forest) with a sequential Monte Carlo tracking mechanism. In so doing, we present the effective application of an anatomical probability map to vessel pruning as well as a supplementary spatial coordinate system for colonic segmentation and registration when this task has been confounded by colon lumen collapse. PMID:25461335

  7. Gearbox fault diagnosis based on deep random forest fusion of acoustic and vibratory signals

    NASA Astrophysics Data System (ADS)

    Li, Chuan; Sanchez, René-Vinicio; Zurita, Grover; Cerrada, Mariela; Cabrera, Diego; Vásquez, Rafael E.

    2016-08-01

    Fault diagnosis is an effective tool to guarantee safe operations in gearboxes. Acoustic and vibratory measurements in such mechanical devices are all sensitive to the existence of faults. This work addresses the use of a deep random forest fusion (DRFF) technique to improve fault diagnosis performance for gearboxes by using measurements of an acoustic emission (AE) sensor and an accelerometer that are used for monitoring the gearbox condition simultaneously. The statistical parameters of the wavelet packet transform (WPT) are first produced from the AE signal and the vibratory signal, respectively. Two deep Boltzmann machines (DBMs) are then developed for deep representations of the WPT statistical parameters. A random forest is finally suggested to fuse the outputs of the two DBMs as the integrated DRFF model. The proposed DRFF technique is evaluated using gearbox fault diagnosis experiments under different operational conditions, and achieves 97.68% of the classification rate for 11 different condition patterns. Compared to other peer algorithms, the addressed method exhibits the best performance. The results indicate that the deep learning fusion of acoustic and vibratory signals may improve fault diagnosis capabilities for gearboxes.

  8. A single amino acid substitution in the hemagglutinin-neuraminidase protein of Newcastle disease virus results in increased fusion and decreased neuraminidase activities without changes in virus pathotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) attachment to the host cell is mediated by the hemagglutinin-neuraminidase (HN), a multifunctional protein that has receptor recognition, neuraminidase and fusion promotion activities. The process that correlates receptor binding and fusion triggering is poorly understo...

  9. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells

    PubMed Central

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K.; Das, Mahua R.; Sen, Shamik; Sarkar, Debi P.; Jana, Siddhartha S.

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (−) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  10. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

    PubMed

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K; Das, Mahua R; Sen, Shamik; Sarkar, Debi P; Jana, Siddhartha S

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  11. Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin.

    PubMed Central

    Kemble, G W; Bodian, D L; Rosé, J; Wilson, I A; White, J M

    1992-01-01

    At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer. Images PMID:1629960

  12. Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide

    SciTech Connect

    Plassmeyer, Matthew L.; Soldan, Samantha S.; Stachelek, Karen M.; Roth, Susan M.; Martin-Garcia, Julio; Gonzalez-Scarano, Francisco . E-mail: scarano@mail.med.upenn.edu

    2007-02-20

    The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

  13. Novel Inhibitors of Influenza Virus Fusion: Structure-Activity Relationship and Interaction with the Viral Hemagglutinin▿

    PubMed Central

    Vanderlinden, Evelien; Göktaş, Fusun; Cesur, Zafer; Froeyen, Matheus; Reed, Mark L.; Russell, Charles J.; Cesur, Nesrin; Naesens, Lieve

    2010-01-01

    A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 μM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors. PMID:20181685

  14. Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion.

    PubMed Central

    Gaudin, Y; Ruigrok, R W; Knossow, M; Flamand, A

    1993-01-01

    Fusion of rabies virus with membranes occurs at acidic pH and is mediated by the viral spike glycoprotein (G). In this paper, we provide the basis for a description of structural transitions associated with exposure to low pH and of their role in membrane fusion. First, we have extended previous studies of fusion kinetics and we have shown that low-pH inhibition of fusion is detectable at 0.5 pH units higher than fusion. Second, low-pH-induced conformational changes were analyzed by using electron microscopy and monoclonal antibody binding assays. The existence of a pH-dependent equilibrium between the native and a low-pH inactive conformation was demonstrated. Third, besides these two conformations, we, using the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid), provide evidence for the existence of a transient third state which appears to be more hydrophobic than the native state. Our results suggest that this transient state is responsible for viral aggregation at low pH and could play a role in the first steps of the fusion mechanism. Images PMID:8437221

  15. Coupling of Human Immunodeficiency Virus Type 1 Fusion to Virion Maturation: a Novel Role of the gp41 Cytoplasmic Tail

    PubMed Central

    Wyma, Donald J.; Jiang, Jiyang; Shi, Jiong; Zhou, Jing; Lineberger, Janet E.; Miller, Michael D.; Aiken, Christopher

    2004-01-01

    Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This “inside-out” regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles. PMID:15016865

  16. Monitoring Forest Carbon Dynamics for REDD: A Landsat-Lidar Fusion Approach

    NASA Astrophysics Data System (ADS)

    Huang, C.; Dubayah, R.; Hurtt, G. C.; Goward, S. N.; Masek, J. G.; Zhu, Z.

    2010-12-01

    Reducing Emissions from Deforestation and Forest Degradation (REDD) is an effort to create a financial value for the carbon stored in forests and to offer incentives for developing countries to reduce emissions from forested lands. Implementing this effort requires methods for quantifying forest carbon and change. Such methods should be accurate enough to allow reliable reporting and efficient enough to enable timely verification and monitoring. Here we present a Landsat-lidar fusion approach for monitoring the dynamics of forest carbon. In this approach, time series Landsat observations are used to detect and date forest disturbance and to track the spectral trajectory of post-disturbance recovery using a vegetation change tracker (VCT) algorithm. Biomass estimates derived from LVIS lidar samples will then be used to establish relationships between standing biomass and age since disturbance and the recovery trajectory. Such relationships can be used to estimate forest biomass not only during the model year, but also for the years after the model year. This is because each disturbance has a time stamp, which can be used to calculate the age since disturbance and the post-disturbance recovery trajectory for any year after the disturbance year using available Landsat images. Therefore, it can not only be used to establish baseline estimates, but also to monitor changes due to both disturbances and recovery. Furthermore, the fine spatial resolutions of the Landsat and LVIS data allow the biomass and biomass change estimates to be derived at hectare or sub-hectare levels. Such fine grain sizes will allow reliable reporting at patch or individual land owner level, which is required for fine scale carbon management and carbon trade at individual land owner level. Critical environmental variables controlling biomass recovery rates may also be revealed by analyzing the variability of age/biomass relationships among patches. The effectiveness of the described approach has

  17. Achieving Accuracy Requirements for Forest Biomass Mapping: A Data Fusion Method for Estimating Forest Biomass and LiDAR Sampling Error with Spaceborne Data

    NASA Technical Reports Server (NTRS)

    Montesano, P. M.; Cook, B. D.; Sun, G.; Simard, M.; Zhang, Z.; Nelson, R. F.; Ranson, K. J.; Lutchke, S.; Blair, J. B.

    2012-01-01

    The synergistic use of active and passive remote sensing (i.e., data fusion) demonstrates the ability of spaceborne light detection and ranging (LiDAR), synthetic aperture radar (SAR) and multispectral imagery for achieving the accuracy requirements of a global forest biomass mapping mission. This data fusion approach also provides a means to extend 3D information from discrete spaceborne LiDAR measurements of forest structure across scales much larger than that of the LiDAR footprint. For estimating biomass, these measurements mix a number of errors including those associated with LiDAR footprint sampling over regional - global extents. A general framework for mapping above ground live forest biomass (AGB) with a data fusion approach is presented and verified using data from NASA field campaigns near Howland, ME, USA, to assess AGB and LiDAR sampling errors across a regionally representative landscape. We combined SAR and Landsat-derived optical (passive optical) image data to identify forest patches, and used image and simulated spaceborne LiDAR data to compute AGB and estimate LiDAR sampling error for forest patches and 100m, 250m, 500m, and 1km grid cells. Forest patches were delineated with Landsat-derived data and airborne SAR imagery, and simulated spaceborne LiDAR (SSL) data were derived from orbit and cloud cover simulations and airborne data from NASA's Laser Vegetation Imaging Sensor (L VIS). At both the patch and grid scales, we evaluated differences in AGB estimation and sampling error from the combined use of LiDAR with both SAR and passive optical and with either SAR or passive optical alone. This data fusion approach demonstrates that incorporating forest patches into the AGB mapping framework can provide sub-grid forest information for coarser grid-level AGB reporting, and that combining simulated spaceborne LiDAR with SAR and passive optical data are most useful for estimating AGB when measurements from LiDAR are limited because they minimized

  18. Discovery of Piperazinylquinoline Derivatives as Novel Respiratory Syncytial Virus Fusion Inhibitors.

    PubMed

    Zheng, Xiufang; Wang, Lisha; Wang, Baoxia; Miao, Kun; Xiang, Kunlun; Feng, Song; Gao, Lu; Shen, Hong C; Yun, Hongying

    2016-06-01

    A novel series of piperazinylquinoline derivatives were discovered as respiratory syncytial virus (RSV) fusion inhibitors by the ligand-based screening approach. Among 3,000 hits, 1-amino-3-[[2-(4-phenyl-1-piperidyl)-4-quinolyl]amino]propan-2-ol (7) was proven to be active against the RSV long (A) strain. The anti-RSV activity was improved by converting piperidine to benzylcarbonyl substituted piperazine. The basic side chain was also found to be crucial for anti-RSV activity. The selected analogues, 45 and 50, demonstrated anti-RSV activities up to EC50 = 0.028 μM and 0.033 μM, respectively. A direct anti-RSV effect was confirmed by a plaque reduction assay and a fusion inhibition assay. Both 45 and 50 showed promising DMPK properties with good oral bioavailability, and could potentially lead to novel therapeutic agents targeting the RSV fusion process. PMID:27326326

  19. Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity

    SciTech Connect

    Roller, Devin G.; Dollery, Stephen J.; Doyle, James L.; Nicola, Anthony V.

    2008-12-20

    Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with emphasis on the cellular receptors for HSV, nectin-1, nectin-2 and HVEM. The ability of an HSV strain with FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral entry by that receptor. A FFWO form of gB was not sufficient to switch the entry of HSV from a pH-dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, the results indicate that the roles of gB in FFWO and in virus-cell fusion during entry are related but not identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity.

  20. Intersubunit disulfide isomerization controls membrane fusion of human T-cell leukemia virus Env.

    PubMed

    Li, Kejun; Zhang, Shujing; Kronqvist, Malin; Wallin, Michael; Ekström, Maria; Derse, David; Garoff, Henrik

    2008-07-01

    Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C(225)XXC, in the C-terminal domain SU. Here we have tested whether this motif is used for isomerization of the intersubunit disulfide of Env and whether this rearrangement is required for membrane fusion. We introduced the C225A and C228A mutations into Env and found that the former but not the latter mutant matured into covalently linked SU-TM complexes in transfected cells. Next, we constructed a secreted Env ectodomain and showed that it underwent incubation-dependent intersubunit disulfide isomerization on target cells. However, the rearrangement was blocked by the C225A mutation, suggesting that C(225) carried the isomerization-active thiol. Still, it was possible to reduce the intersubunit disulfide of the native C225A ectodomain mutant with dithiothreitol (DTT). The importance of the CXXC-mediated disulfide isomerization for infection was studied using murine leukemia virus vectors pseudotyped with wild-type or C225A HTLV-1 Env. We found that the mutant Env blocked infection, but this could be rescued with DTT. The fusion activity was tested in a fusion-from-within assay using a coculture of rat XC target and transfected BHK-21 effector cells. We found that the mutation blocked polykaryon formation, but this could be reversed with DTT. Similar DTT-reversible inhibition of infection and fusion was observed when a membrane-impermeable alkylator was present during the infection/fusion incubation. We conclude that the fusion activity of HTLV-1 Env is controlled by an SU CXXC-mediated isomerization of the intersubunit disulfide. Thus, this extends the applicability of the isomerization model from gammaretroviruses to deltaretroviruses. PMID:18480461

  1. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  2. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  3. Phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein DAS181

    PubMed Central

    Triana-Baltzer, Gallen B.; Sanders, Rebecca L.; Hedlund, Maria; Jensen, Kellie A.; Aschenbrenner, Laura M.; Larson, Jeffrey L.; Fang, Fang

    2011-01-01

    Background Influenza viruses (IFVs) frequently achieve resistance to antiviral drugs, necessitating the development of compounds with novel mechanisms of action. DAS181 (Fludase®), a sialidase fusion protein, may have a reduced potential for generating drug resistance due to its novel host-targeting mechanism of action. Methods IFV strains B/Maryland/1/59 and A/Victoria/3/75 (H3N2) were subjected to >30 passages under increasing selective pressure with DAS181. The DAS181-selected IFV isolates were characterized in vitro and in mice. Results Despite extensive passaging, DAS181-selected viruses exhibited a very low level of resistance to DAS181, which ranged between 3- and 18-fold increase in EC50. DAS181-selected viruses displayed an attenuated phenotype in vitro, as exhibited by slower growth, smaller plaque size and increased particle to pfu ratios relative to wild-type virus. Further, the DAS181 resistance phenotype was unstable and was substantially reversed over time upon DAS181 withdrawal. In mice, the DAS181-selected viruses exhibited no greater virulence than their wild-type counterparts. Genotypic and phenotypic analysis of DAS181-selected viruses revealed mutations in the haemagglutinin (HA) and neuraminidase (NA) molecules and also changes in HA and NA function. Conclusions Results indicate that resistance to DAS181 is minimal and unstable. The DAS181-selected IFV isolates exhibit reduced fitness in vitro, likely due to altered HA and NA functions. PMID:21097900

  4. Avian sarcoma and leukosis virus-receptor interactions: From classical genetics to novel insights into virus-cell membrane fusion

    SciTech Connect

    Barnard, R.J.O.; Elleder, D.; Young, J.A.T. . E-mail: jyoung@salk.edu

    2006-01-05

    For over 40 years, avian sarcoma and leukosis virus (ASLV)-receptor interactions have been employed as a useful model system to study the mechanism of retroviral entry into cells. Pioneering studies on this system focused upon the genetic basis of the differential susceptibilities of different lines of chickens to infection by distinct subgroups of ASLV. These studies led to the definition of three distinct autosomal recessive genes that were predicted to encode cellular receptors for different viral subgroups. They also led to the concept of viral interference, i.e. the mechanism by which infection by one virus can render cells resistant to reinfection by other viruses that use the same cellular receptor. Here, we review the contributions that analyses of the ASLV-receptor system have made in unraveling the mechanisms of retroviral entry into cells and focus on key findings such as identification and characterization of the ASLV receptor genes and the subsequent elucidation of an unprecedented mechanism of virus-cell fusion. Since many of the initial findings on this system were published in the early volumes of Virology, this subject is especially well suited to this special anniversary issue of the journal.

  5. Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells.

    PubMed

    Arrigo, S J; Haines, J K; Huffman, K M

    1995-01-01

    We have generated various mammalian expression constructs that produce fusion proteins of human immunodeficiency virus type 1 (HIV-1) protease (PR) with the HIV-1 Nef protein. The expression of these proteins is inducible by the HIV-1 Tat protein. High-level expression of proteolytically active PR was produced from PR imbedded into Nef coding sequences, flanked by PR cleavage sites. The fusion protein was cleaved nearly to completion and did not exhibit the regulated processing that is seen with the virally encoded PR. No cytotoxic effect of PR expression was detected. The self-cleavage of PR could be inhibited by a specific inhibitor of HIV-1 PR (U75875). Elimination of the aminoterminal PR cleavage site did not have a measurable effect on cleavage of the precursor fusion protein. The cleaved fusion proteins appeared to be extremely unstable in the transfected cells. These findings demonstrate the intrinsic activity of HIV-1 PR in mammalian cells, in the context of a heterologous fusion protein. PMID:7832989

  6. C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2006-12-20

    Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles.

  7. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion*

    PubMed Central

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  8. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    PubMed

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  9. Crystal structure of the conserved herpes virus fusion regulator complex gH-gL

    SciTech Connect

    Chowdary, Tirumala K; Cairns, Tina M; Atanasiu, Doina; Cohen, Gary H; Eisenberg, Roselyn J; Heldwein, Ekaterina E

    2010-09-13

    Herpesviruses, which cause many incurable diseases, infect cells by fusing viral and cellular membranes. Whereas most other enveloped viruses use a single viral catalyst called a fusogen, herpesviruses, inexplicably, require two conserved fusion-machinery components, gB and the heterodimer gH-gL, plus other nonconserved components. gB is a class III viral fusogen, but unlike other members of its class, it does not function alone. We determined the crystal structure of the gH ectodomain bound to gL from herpes simplex virus 2. gH-gL is an unusually tight complex with a unique architecture that, unexpectedly, does not resemble any known viral fusogen. Instead, we propose that gH-gL activates gB for fusion, possibly through direct binding. Formation of a gB-gH-gL complex is critical for fusion and is inhibited by a neutralizing antibody, making the gB-gH-gL interface a promising antiviral target.

  10. Crystal Structure of the Conserved Herpes Virus Fusion Regulator Complex gH–gL

    SciTech Connect

    Chowdary, T.; Cairns, T; Atanasiu, D; Cohen, G; Eisenberg, R; Heldwein, E

    2010-01-01

    Herpesviruses, which cause many incurable diseases, infect cells by fusing viral and cellular membranes. Whereas most other enveloped viruses use a single viral catalyst called a fusogen, herpesviruses, inexplicably, require two conserved fusion-machinery components, gB and the heterodimer gH-gL, plus other nonconserved components. gB is a class III viral fusogen, but unlike other members of its class, it does not function alone. We determined the crystal structure of the gH ectodomain bound to gL from herpes simplex virus 2. gH-gL is an unusually tight complex with a unique architecture that, unexpectedly, does not resemble any known viral fusogen. Instead, we propose that gH-gL activates gB for fusion, possibly through direct binding. Formation of a gB-gH-gL complex is critical for fusion and is inhibited by a neutralizing antibody, making the gB-gH-gL interface a promising antiviral target.

  11. A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion.

    PubMed

    Shi, Xin; Deng, Yongqiang; Wang, Huajing; Ji, Guanghui; Tan, Wenlong; Jiang, Tao; Li, Xiaofeng; Zhao, Hui; Xia, Tian; Meng, Yanchun; Wang, Chao; Yu, Xiaojie; Yang, Yang; Li, Bohua; Qin, E-De; Dai, Jianxin; Qin, Cheng-Feng; Guo, Yajun

    2016-01-01

    Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV. PMID:26905804

  12. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    SciTech Connect

    Bernard, Eric; Simmons, Graham; Chazal, Nathalie; and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  13. Remote sensing based detection of forested wetlands: An evaluation of LiDAR, aerial imagery, and their data fusion

    NASA Astrophysics Data System (ADS)

    Suiter, Ashley Elizabeth

    Multi-spectral imagery provides a robust and low-cost dataset for assessing wetland extent and quality over broad regions and is frequently used for wetland inventories. However in forested wetlands, hydrology is obscured by tree canopy making it difficult to detect with multi-spectral imagery alone. Because of this, classification of forested wetlands often includes greater errors than that of other wetlands types. Elevation and terrain derivatives have been shown to be useful for modelling wetland hydrology. But, few studies have addressed the use of LiDAR intensity data detecting hydrology in forested wetlands. Due the tendency of LiDAR signal to be attenuated by water, this research proposed the fusion of LiDAR intensity data with LiDAR elevation, terrain data, and aerial imagery, for the detection of forested wetland hydrology. We examined the utility of LiDAR intensity data and determined whether the fusion of Lidar derived data with multispectral imagery increased the accuracy of forested wetland classification compared with a classification performed with only multi-spectral image. Four classifications were performed: Classification A -- All Imagery, Classification B -- All LiDAR, Classification C -- LiDAR without Intensity, and Classification D -- Fusion of All Data. These classifications were performed using random forest and each resulted in a 3-foot resolution thematic raster of forested upland and forested wetland locations in Vermilion County, Illinois. The accuracies of these classifications were compared using Kappa Coefficient of Agreement. Importance statistics produced within the random forest classifier were evaluated in order to understand the contribution of individual datasets. Classification D, which used the fusion of LiDAR and multi-spectral imagery as input variables, had moderate to strong agreement between reference data and classification results. It was found that Classification A performed using all the LiDAR data and its derivatives

  14. A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus-cell fusion.

    PubMed Central

    Kumar, M; Hassan, M Q; Tyagi, S K; Sarkar, D P

    1997-01-01

    Sendai virus envelopes devoid of hemagglutinin-neuraminidase but containing the fusion protein (F-virosomes) were prepared. F-virosomes exhibited discernible serine protease activity at neutral pH. Electrophoretic analysis of the protein profile of the F-virosomes under nonreducing conditions, by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, led to the identification of a previously unknown glycoprotein with a relative molecular weight of 45,000 (45K protein) associated with the F protein. The identity of the 45K protein, as distinct from F protein, was established by Western blot analysis with F- and 45K-specific antibodies. This 45K protein forms a nexus with the F protein through noncovalent hydrophobic interactions, as proved by its sensitivity to urea treatment, and it is essential for the proteolytic activity of the F-virosomes as well as for the fusion of the viral envelope with host cell membrane. N-terminal sequence analysis (first 11 amino acids) of this protein showed strong homology (> 90%) to flavivirus NS3 serine proteases but no similarity to any of the Sendai viral proteins. On the basis of the N-terminal sequence, oligonucleotides were designed corresponding to the sense and antisense DNA sequences. Dot blot hybridization and primer extension with these oligonucleotides with the viral and the host genome confirmed the host origin of this protein. Further, the limited proteolytic digestion of the target membrane resulted in significant inhibition of viral fusion with it. On the basis of these results, we postulate a model for the molecular mechanism of F protein-induced membrane fusion, which may provide a rationale for other paramyxoviruses. PMID:9261357

  15. Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

    PubMed Central

    Khosravi, Mojtaba; Bringolf, Fanny; Röthlisberger, Silvan; Bieringer, Maria; Schneider-Schaulies, Jürgen; Zurbriggen, Andreas; Origgi, Francesco

    2015-01-01

    ABSTRACT Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces (“front” and “back”). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. IMPORTANCE A complete understanding of the measles virus

  16. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

    PubMed Central

    Zheng, Yi-Min; Melikyan, Gregory B.; Liu, Shan-Lu; Cohen, Fredric S.

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  17. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

    PubMed

    Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  18. Structural Characterization of an Early Fusion Intermediate of Influenza Virus Hemagglutinin

    SciTech Connect

    Xu, Rui; Wilson, Ian A.

    2011-12-07

    The hemagglutinin (HA) envelope protein of influenza virus mediates viral entry through membrane fusion in the acidic environment of the endosome. Crystal structures of HA in pre- and postfusion states have laid the foundation for proposals for a general fusion mechanism for viral envelope proteins. The large-scale conformational rearrangement of HA at low pH is triggered by a loop-to-helix transition of an interhelical loop (B loop) within the fusion domain and is often referred to as the 'spring-loaded' mechanism. Although the receptor-binding HA1 subunit is believed to act as a 'clamp' to keep the B loop in its metastable prefusion state at neutral pH, the 'pH sensors' that are responsible for the clamp release and the ensuing structural transitions have remained elusive. Here we identify a mutation in the HA2 fusion domain from the influenza virus H2 subtype that stabilizes the HA trimer in a prefusion-like state at and below fusogenic pH. Crystal structures of this putative early intermediate state reveal reorganization of ionic interactions at the HA1-HA2 interface at acidic pH and deformation of the HA1 membrane-distal domain. Along with neutralization of glutamate residues on the B loop, these changes cause a rotation of the B loop and solvent exposure of conserved phenylalanines, which are key residues at the trimer interface of the postfusion structure. Thus, our study reveals the possible initial structural event that leads to release of the B loop from its prefusion conformation, which is aided by unexpected structural changes within the membrane-distal HA1 domain at low pH.

  19. Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

    PubMed Central

    Xu, Rui; Palmer, Samantha G.; Porotto, Matteo; Palermo, Laura M.; Niewiesk, Stefan; Wilson, Ian A.; Moscona, Anne

    2013-01-01

    ABSTRACT Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by

  20. A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

    PubMed Central

    Hollmann, Axel; Tanner, Lukas B.; Akyol Ataman, Zeynep; Yun, Tatyana; Shui, Guanghou; Aguilar, Hector C.; Zhang, Dong; Meriwether, David; Roman-Sosa, Gleyder; Robinson, Lindsey R.; Juelich, Terry L.; Buczkowski, Hubert; Chou, Sunwen; Castanho, Miguel A. R. B.; Wolf, Mike C.; Smith, Jennifer K.; Banyard, Ashley; Kielian, Margaret; Reddy, Srinivasa; Wenk, Markus R.; Selke, Matthias; Santos, Nuno C.; Freiberg, Alexander N.; Jung, Michael E.; Lee, Benhur

    2013-01-01

    LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (1O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated 1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. 1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced 1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of 1O2 generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the

  1. Different Regions of the Newcastle Disease Virus Fusion Protein Modulate Pathogenicity

    PubMed Central

    Heiden, Sandra; Grund, Christian; Röder, Anja; Granzow, Harald; Kühnel, Denis; Mettenleiter, Thomas C.; Römer-Oberdörfer, Angela

    2014-01-01

    Newcastle disease virus (NDV), also designated as Avian paramyxovirus type 1 (APMV-1), is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F) is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic), intermediate (mesogenic) and low (lentogenic) virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1) isolate with an intracerebral pathogenicity index (ICPI) of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site. PMID:25437176

  2. Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

    PubMed Central

    Nakahashi, Mito; Matsushima, Yoshiaki; Ito, Morihiro; Nishio, Machiko; Kawano, Mitsuo; Komada, Hiroshi; Nosaka, Tetsuya

    2013-01-01

    For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity. PMID:23698295

  3. Constructing seasonal LAI trajectory by data-model fusion for global evergreen needle-leaf forests

    NASA Astrophysics Data System (ADS)

    Wang, R.; Chen, J.; Mo, G.

    2010-12-01

    For decades, advancements in optical remote sensors made it possible to produce maps of a biophysical parameter--the Leaf Area Index (LAI), which is critically necessary in regional and global modeling of exchanges of carbon, water, energy and other substances, across large areas in a fast way. Quite a few global LAI products have been generated since 2000, e.g. GLOBCARBON (Deng et al., 2006), MODIS Collection 5 (Shabanov et al., 2007), CYCLOPES (Baret et al., 2007), etc. Albeit these progresses, the basic physics behind the technology restrains it from accurate estimation of LAI in winter, especially for northern high-latitude evergreen needle-leaf forests. Underestimation of winter LAI in these regions has been reported in literature (Yang et al., 2000; Cohen et al., 2003; Tian et al., 2004; Weiss et al., 2007; Pisek et al., 2007), and the distortion is usually attributed to the variations of canopy reflectance caused by understory change (Weiss et al., 2007) as well as by the presence of ice and snow on leaves and ground (Cohen, 2003; Tian et al., 2004). Seasonal changes in leaf pigments can also be another reason for low LAI retrieved in winter. Low conifer LAI values in winter retrieved from remote sensing make them unusable for surface energy budget calculations. To avoid these drawbacks of remote sensing approaches, we attempt to reconstruct the seasonal LAI trajectory through model-data fusion. A 1-degree LAI map of global evergreen needle-leaf forests at 10-day interval is produced based on the carbon allocation principle in trees. With net primary productivity (NPP) calculated by the Boreal Ecosystems Productivity Simulator (BEPS) (Chen et al., 1999), carbon allocated to needles is quantitatively evaluated and then can be further transformed into LAI using the specific leaf area (SLA). A leaf-fall scheme is developed to mimic the carbon loss caused by falling needles throughout the year. The seasonally maximum LAI from remote sensing data for each pixel

  4. Effect of the ionophore monensin on herpes simplex virus type 1-induced cell fusion, glycoprotein synthesis, and virion infectivity.

    PubMed

    Kousoulas, K G; Bzik, D J; Person, S

    1983-01-01

    The ionophore monensin inhibited the formation of mature, fully glycosylated glycoproteins gB, gC, and gD during herpes simplex virus type 1 infection of human embryonic lung cells. Underglycosylated forms, including the apparent high-mannose precursor forms of the major glycoproteins, appeared. Monensin inhibited virus-induced cell fusion. Infectious virions produced in the presence of monensin appeared to contain predominantly underglycosylated glycoproteins. PMID:6307921

  5. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  6. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

    SciTech Connect

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2011-09-16

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.

  7. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    SciTech Connect

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and

  8. Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

    PubMed Central

    Lulla, Aleksei; Lulla, Valeria; Tints, Kairit; Ahola, Tero; Merits, Andres

    2006-01-01

    The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1′ had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1′, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site. PMID:16699022

  9. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

    SciTech Connect

    Yu, I.-M.; Holdaway, H.A.; Chipman, P.R.; Kuhn, R.J.; Rossmann, M.G.; Chen, J.; Purdue

    2010-07-27

    Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.

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

    PubMed

    Veazey, Ronald S; Klasse, Per Johan; Schader, Susan M; Hu, Qinxue; Ketas, Thomas J; Lu, Min; Marx, Preston A; Dufour, Jason; Colonno, Richard J; Shattock, Robin J; Springer, Martin S; Moore, John P

    2005-11-01

    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. PMID:16258536

  11. Secretion of the respiratory syncytial virus fusion protein from insect cells using the baculovirus expression system.

    PubMed

    Tan, Boon-Huan; Brown, Gaie; Sugrue, Richard J

    2007-01-01

    Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembrane domain was removed (GST-F2/F1), were cloned into the baculovirus pAcSecG2T secretory vector. These virus sequences also had the endogenous virus signal sequence removed and replaced with a signal sequence derived from the baculovirus gp67 glycoprotein, which was present in pAcSecG2T. The recombinant RSV glycoproteins were successfully detected in expressing cells by immunofluorescence assay and in the tissue culture medium by western blot analysis. The secreted recombinant GST-F2/F1 protein was further analysed using glycosidases. Our results showed that the GST-F2/F1 protein were sensitive to peptide:N-glycosidase F (PNGase F) treatment, but not to Endoglycosidase H (EndoH) treatment. This indicates that the secreted recombinant proteins were modified by the addition of mature N-linked glycan chains. PMID:17502677

  12. A virus causes cancer by inducing massive chromosomal instability through cell fusion.

    PubMed

    Duelli, Dominik M; Padilla-Nash, Hesed M; Berman, David; Murphy, Kathleen M; Ried, Thomas; Lazebnik, Yuri

    2007-03-01

    Chromosomal instability (CIN) underlies malignant properties of many solid cancers and their ability to escape therapy, and it might itself cause cancer [1, 2]. CIN is sustained by deficiencies in proteins, such as the tumor suppressor p53 [3-5], that police genome integrity, but the primary cause of CIN in sporadic cancers remains uncertain [6, 7]. The primary suspects are mutations that deregulate telomere maintenance, or mitosis, yet such mutations have not been identified in the majority of sporadic cancers [6]. Alternatively, CIN could be caused by a transient event that destabilizes the genome without permanently affecting mechanisms of mitosis or proliferation [5, 8]. Here, we show that an otherwise harmless virus rapidly causes massive chromosomal instability by fusing cells whose cell cycle is deregulated by oncogenes. This synergy between fusion and oncogenes "randomizes" normal diploid human fibroblasts so extensively that each analyzed cell has a unique karyotype, and some produce aggressive, highly aneuploid, heterogeneous, and transplantable epithelial cancers in mice. Because many viruses are fusogenic, this study suggests that viruses, including those that have not been linked to carcinogenesis, can cause chromosomal instability and, consequently, cancer by fusing cells. PMID:17320392

  13. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.

    PubMed

    Stewart-Jones, Guillaume B E; Thomas, Paul V; Chen, Man; Druz, Aliaksandr; Joyce, M Gordon; Kong, Wing-Pui; Sastry, Mallika; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S; McLellan, Jason S; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R; Graham, Barney S; Kwong, Peter D

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. PMID:26098893

  14. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

    PubMed Central

    Stewart-Jones, Guillaume B. E.; Thomas, Paul V.; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S.; McLellan, Jason S.; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. PMID:26098893

  15. Is the optimal pH for membrane fusion in host cells by avian influenza viruses related to host range and pathogenicity?

    PubMed

    Okamatsu, Masatoshi; Motohashi, Yurie; Hiono, Takahiro; Tamura, Tomokazu; Nagaya, Kazuki; Matsuno, Keita; Sakoda, Yoshihiro; Kida, Hiroshi

    2016-08-01

    Influenza viruses isolated from wild ducks do not replicate in chickens. This fact is not explained solely by the receptor specificity of the hemagglutinin (HA) from such viruses for target host cells. To investigate this restriction in host range, the fusion activities of HA molecules from duck and chicken influenza viruses were examined. Influenza viruses A/duck/Mongolia/54/2001 (H5N2) (Dk/MNG) and A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR), which replicate only in their primary hosts, were used. The optimal pH for membrane fusion of Ck/IBR was 5.9, higher than that of Dk/MNG at 4.9. To assess the relationship between the optimal pH for fusion and the host range of avian influenza viruses, the optimal pH for fusion of 55 influenza virus strains isolated from ducks and chickens was examined. No correlation was found between the host range and optimal pH for membrane fusion by the viruses, and this finding applied also to the H5N1 highly pathogenic avian influenza viruses. The optimal pH for membrane fusion for avian influenza viruses was shown to not necessarily be correlated with their host range or pathogenicity in ducks and chickens. PMID:27231009

  16. Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

    PubMed

    Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

    2016-07-01

    Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. PMID:27226547

  17. Fusion

    NASA Astrophysics Data System (ADS)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  18. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    SciTech Connect

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  19. Identification of a Potent and Broad-Spectrum Hepatitis C Virus Fusion Inhibitory Peptide from the E2 Stem Domain

    PubMed Central

    Chi, Xiaojing; Niu, Yuqiang; Cheng, Min; Liu, Xiuying; Feng, Yetong; Zheng, Fuxiang; Fan, Jingjing; Li, Xiang; Jin, Qi; Zhong, Jin; Li, Yi-Ping; Yang, Wei

    2016-01-01

    Hepatitis C virus (HCV) envelope proteins E1 and E2 play an essential role in virus entry. However, the fusion mechanisms of HCV remain largely unclear, hampering the development of efficient fusion inhibitors. Here, we developed two cell-based membrane fusion models that allow for screening a peptide library covering the full-length E1 and E2 amino acid sequences. A peptide from the E2 stem domain, named E27, was found to possess the ability to block E1E2-mediated cell-cell fusion and inhibit cell entry of HCV pseudoparticles and infection of cell culture-derived HCV at nanomolar concentrations. E27 demonstrated broad-spectrum inhibition of the major genotypes 1 to 6. A time-of-addition experiment revealed that E27 predominantly functions in the late steps during HCV entry, without influencing the expression and localization of HCV co-receptors. Moreover, we demonstrated that E27 interfered with hetero-dimerization of ectopically expressed E1E2 in cells, and mutational analysis suggested that E27 might target a conserved region in E1. Taken together, our findings provide a novel candidate as well as a strategy for developing potent and broad-spectrum HCV fusion inhibitors, which may complement the current direct-acting antiviral medications for chronic hepatitis C, and shed light on the mechanism of HCV membrane fusion. PMID:27121372

  20. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D.

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  1. Comparative Pathogenesis of Alkhumra Hemorrhagic Fever and Kyasanur Forest Disease Viruses in a Mouse Model

    PubMed Central

    Sawatsky, Bevan; McAuley, Alexander J.; Holbrook, Michael R.; Bente, Dennis A.

    2014-01-01

    Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses. PMID:24922308

  2. Structure of Dengue Virus: Implications for Flavivirus Organization, Maturation, and Fusion

    PubMed Central

    Kuhn, Richard J.; Zhang, Wei; Rossmann, Michael G.; Pletnev, Sergei V.; Corver, Jeroen; Lenches, Edith; Jones, Christopher T.; Mukhopadhyay, Suchetana; Chipman, Paul R.; Strauss, Ellen G.; Baker, Timothy S.; Strauss, James H.

    2014-01-01

    Summary The first structure of a flavivirus has been determined by using a comThe first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal β barrels of domain II of the glycoprotein E are inserted into the cellular membrane. PMID:11893341

  3. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    NASA Astrophysics Data System (ADS)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  4. In vitro and in vivo broad antiviral activity of peptides homologous to fusion glycoproteins of Newcastle disease virus and Marek's disease virus.

    PubMed

    Chi, Xiao-Jing; Wang, Xiao-Jun; Wang, Cheng-Yu; Cui, Xiao-Jing; Wang, Xiao-Jia

    2014-04-01

    Newcastle disease virus (NDV) of paramyxovirus and Marek's disease virus (MDV) of herpesvirus, two of the most serious threats to the poultry industry, can give rise to complex co-infections that hinder diagnosis and prevention. In the current study, two different peptides, derived from the MDV gH (gHH2L) and gB (gBH3), respectively, exhibit antiviral activity against NDV in vitro. The potent inhibitory effect of heptad repeat 2 from fusion glycoprotein of the NDV on MDV infection also has been demonstrated. Plaque formation and embryo infectivity assays confirmed these antiviral results. Furthermore, each tandem peptide consisting of two motifs from different viruses exhibits more potent antiviral activity than the constituent peptides. The current work provides a new strategy for developing novel peptides and vaccines against virus infection and co-infections. PMID:24412629

  5. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    PubMed

    Hernáez, Bruno; Guerra, Milagros; Salas, María L; Andrés, Germán

    2016-04-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  6. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  7. Automated segmentation of thyroid gland on CT images with multi-atlas label fusion and random classification forest

    NASA Astrophysics Data System (ADS)

    Liu, Jiamin; Chang, Kevin; Kim, Lauren; Turkbey, Evrim; Lu, Le; Yao, Jianhua; Summers, Ronald

    2015-03-01

    The thyroid gland plays an important role in clinical practice, especially for radiation therapy treatment planning. For patients with head and neck cancer, radiation therapy requires a precise delineation of the thyroid gland to be spared on the pre-treatment planning CT images to avoid thyroid dysfunction. In the current clinical workflow, the thyroid gland is normally manually delineated by radiologists or radiation oncologists, which is time consuming and error prone. Therefore, a system for automated segmentation of the thyroid is desirable. However, automated segmentation of the thyroid is challenging because the thyroid is inhomogeneous and surrounded by structures that have similar intensities. In this work, the thyroid gland segmentation is initially estimated by multi-atlas label fusion algorithm. The segmentation is refined by supervised statistical learning based voxel labeling with a random forest algorithm. Multiatlas label fusion (MALF) transfers expert-labeled thyroids from atlases to a target image using deformable registration. Errors produced by label transfer are reduced by label fusion that combines the results produced by all atlases into a consensus solution. Then, random forest (RF) employs an ensemble of decision trees that are trained on labeled thyroids to recognize features. The trained forest classifier is then applied to the thyroid estimated from the MALF by voxel scanning to assign the class-conditional probability. Voxels from the expert-labeled thyroids in CT volumes are treated as positive classes; background non-thyroid voxels as negatives. We applied this automated thyroid segmentation system to CT scans of 20 patients. The results showed that the MALF achieved an overall 0.75 Dice Similarity Coefficient (DSC) and the RF classification further improved the DSC to 0.81.

  8. Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus.

    PubMed Central

    Zhou, X; Berglund, P; Zhao, H; Liljeström, P; Jondal, M

    1995-01-01

    The Semliki Forest virus (SFV) expression system can be used to package recombinant RNA into infectious suicide particles. Such RNA encodes only the SFV replicase and the heterologous protein but no structural proteins of SFV, and it is thus deficient in productive replication. We demonstrate here that infection of C57BL/6 (H-2b) and BALB/c (H-2d) mice with recombinant SFV expressing the nucleoprotein (NP) of influenza virus (SFV-NP) resulted in efficient priming of influenza virus-specific CD8+ cytotoxic T-cell (CTL) responses. The generated CTLs lysed both homologous (A/PR/8/34) and heterologous (A/HK/68) influenza virus-infected, or peptide-coated, target cells to a similar degree as CTLs induced by wild-type influenza virus in a major histocompatibility complex class I-restricted fashion. As few as 100 infectious units of virus induced a strong CTL response. Induction of CTL by SFV-NP could also be achieved in CD4 gene-targeted mice, demonstrating the independence of the primary CTL response of CD4+ helper T cells. One immunization generated a CTL response that peaked after 1 week, and an additional booster injection generated a CTL memory, which was still detectable after 40 days. SFV-NP immunizations also generated high-titered IgG humoral responses that remained significant after several months. These results demonstrate that the recombinant SFV suicide system is highly efficient in antigen presentation and suggest that it may have a potential as a recombinant vaccine. Images Fig. 1 PMID:7708765

  9. Template RNA Length Determines the Size of Replication Complex Spherules for Semliki Forest Virus

    PubMed Central

    Kallio, Katri; Hellström, Kirsi; Balistreri, Giuseppe; Spuul, Pirjo; Jokitalo, Eija

    2013-01-01

    The replication complexes of positive-strand RNA viruses are always associated with cellular membranes. The morphology of the replication-associated membranes is altered in different ways in different viral systems, but many viruses induce small membrane invaginations known as spherules as their replication sites. We show here that for Semliki Forest virus (SFV), an alphavirus, the size of the spherules is tightly connected with the length of the replicating RNA template. Cells with different model templates, expressed in trans and copied by the viral replicase, were analyzed with correlative light and electron microscopy. It was demonstrated that the viral-genome-sized template of 11.5 kb induced spherules that were ∼58 nm in diameter, whereas a template of 6 kb yielded ∼39-nm spherules. Different sizes of viral templates were replicated efficiently in trans, as assessed by radioactive labeling and Northern blotting. The replication of two different templates, in cis and trans, yielded two size classes of spherules in the same cell. These results indicate that RNA plays a crucial determining role in spherule assembly for SFV, in direct contrast with results from other positive-strand RNA viruses, in which either the presence of viral RNA or the RNA size do not contribute to spherule formation. PMID:23760239

  10. Replication competent Semliki Forest virus prolongs survival in experimental lung cancer.

    PubMed

    Määttä, Ann-Marie; Mäkinen, Kimmo; Ketola, Anna; Liimatainen, Timo; Yongabi, Felicitas Newu; Vähä-Koskela, Markus; Pirinen, Risto; Rautsi, Outi; Pellinen, Riikka; Hinkkanen, Ari; Wahlfors, Jarmo

    2008-10-01

    We evaluated the therapeutic potential of the replication competent vector VA7-EGFP, which is based on the avirulent Semliki Forest virus (SFV) strain A7 (74) carrying the EGFP marker gene in an orthotopic lung cancer tumor model in nude mice. We have previously shown that this oncolytic vector destroys tumor cells efficiently in vitro and in vivo (in subcutaneous tumor model). Tumor growth in animals with orthotopically implanted adenocarcinoma cells (A549) were monitored during the study with small animal CT. We show that locally administered virotherapy with VA7-EGFP increased survival rate in experimental lung cancer significantly (p < 0.001) comparable to results obtained with the second generation conditionally replicating adenoviral vector Ad5-Delta24TK-GFP, used for comparison. The limited efficacy in systemically administered oncolytic viruses is the essential problem in oncolytic virotherapy and also in this study we were not able to elicit significant response with systemic administration route. Despite the fact that tumor microenvironment in orthotopic lung cancer is more optimal, viruses failed to home to the tumors and were unable to initiate efficient intratumoral replication. Clearly, the efficacy of virotherapy is influenced by many factors such as the route of virus administration, immunological and physiological barriers and cancer cell-specific features (IFN-responsiveness). PMID:18651570

  11. Viruses accumulate in aging infection centers of a fungal forest pathogen

    PubMed Central

    Vainio, Eeva J; Müller, Michael M; Korhonen, Kari; Piri, Tuula; Hantula, Jarkko

    2015-01-01

    Fungal viruses (mycoviruses) with RNA genomes are believed to lack extracellular infective particles. These viruses are transmitted laterally among fungal strains through mycelial anastomoses or vertically via their infected spores, but little is known regarding their prevalence and patterns of dispersal under natural conditions. Here, we examined, in detail, the spatial and temporal changes in a mycovirus community and its host fungus Heterobasidion parviporum, the most devastating fungal pathogen of conifers in the Boreal forest region. During the 7-year sampling period, viruses accumulated in clonal host individuals as a result of indigenous viruses spreading within and between clones as well as novel strains arriving via airborne spores. Viral community changes produced pockets of heterogeneity within large H. parviporum clones. The appearance of novel viral infections in aging clones indicated that transient cell-to-cell contacts between Heterobasidion strains are likely to occur more frequently than what was inferred from genotypic analyses. Intraspecific variation was low among the three partitivirus species at the study site, whereas the unassigned viral species HetRV6 was highly polymorphic. The accumulation of point mutations during persistent infections resulted in viral diversification, that is, the presence of nearly identical viral sequence variants within single clones. Our results also suggest that co-infections by distantly related viral species are more stable than those between conspecific strains, and mutual exclusion may play a role in determining mycoviral communities. PMID:25126757

  12. Development of a subgenomic clone system for Kyasanur Forest disease virus.

    PubMed

    Cook, Bradley W M; Nikiforuk, Aidan M; Cutts, Todd A; Kobasa, Darwyn; Court, Deborah A; Theriault, Steven S

    2016-07-01

    Emerging tropical viruses pose an increasing threat to public health because social, economic and environmental factors such as global trade and deforestation allow for their migration into previously unexposed populations and ecological niches. Among such viruses, Kyasanur Forest disease virus (KFDV) deserves particular recognition because it causes hemorrhagic fever. This work describes the completion of an antiviral testing platform (subgenomic system) for KFDV that could be used to quickly and safely screen compounds capable of inhibiting KFDV replication without the requirement for high containment, as the structural genes have been replaced with a luciferase reporter gene precluding the generation of infectious particles. The coordination of KFDV kinetics with the replication characteristics of the subgenomic system has provided additional insight into the timing of flavivirus replication events, as the genetically engineered KFDV genome began replication as early as 2h post cellular entry. Possession of such antiviral testing platforms by public health agencies should accelerate the testing of antiviral drugs against emerging or recently emerged viruses mitigating the effects of their disease and transmission. PMID:27357207

  13. Inhibition of Nipah Virus Infectin In Vivo: Targeting an Early Stage of Paramyxovirus Fusion Activation during Viral Entry

    SciTech Connect

    M Porotto; B Rockx; C Yokoyama; A Talekar; I DeVito; l Palermo; J Liu; R Cortese; M Lu; et al.

    2011-12-31

    In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.

  14. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  15. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus.

    PubMed

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  16. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

    PubMed Central

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  17. A Structurally Unresolved Head Segment of Defined Length Favors Proper Measles Virus Hemagglutinin Tetramerization and Efficient Membrane Fusion Triggering

    PubMed Central

    Navaratnarajah, Chanakha K.; Rosemarie, Quincy

    2015-01-01

    ABSTRACT Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers. IMPORTANCE Understanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion

  18. N-Glycans on the Nipah Virus Attachment Glycoprotein Modulate Fusion and Viral Entry as They Protect against Antibody Neutralization

    PubMed Central

    Biering, Scott B.; Huang, Andrew; Vu, Andy T.; Robinson, Lindsey R.; Bradel-Tretheway, Birgit; Choi, Eric

    2012-01-01

    Nipah virus (NiV) is the deadliest known paramyxovirus. Membrane fusion is essential for NiV entry into host cells and for the virus' pathological induction of cell-cell fusion (syncytia). The mechanism by which the attachment glycoprotein (G), upon binding to the cell receptors ephrinB2 or ephrinB3, triggers the fusion glycoprotein (F) to execute membrane fusion is largely unknown. N-glycans on paramyxovirus glycoproteins are generally required for proper protein conformational integrity, transport, and sometimes biological functions. We made conservative mutations (Asn to Gln) at the seven potential N-glycosylation sites in the NiV G ectodomain (G1 to G7) individually or in combination. Six of the seven N-glycosylation sites were found to be glycosylated. Moreover, pseudotyped virions carrying these N-glycan mutants had increased antibody neutralization sensitivities. Interestingly, our results revealed hyperfusogenic and hypofusogenic phenotypes for mutants that bound ephrinB2 at wild-type levels, and the mutant's cell-cell fusion phenotypes generally correlated to viral entry levels. In addition, when removing multiple N-glycans simultaneously, we observed synergistic or dominant-negative membrane fusion phenotypes. Interestingly, our data indicated that 4- to 6-fold increases in fusogenicity resulted from multiple mechanisms, including but not restricted to the increase of F triggering. Altogether, our results suggest that NiV-G N-glycans play a role in shielding virions against antibody neutralization, while modulating cell-cell fusion and viral entry via multiple mechanisms. PMID:22915812

  19. Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

    PubMed Central

    Kanaseki, Toku; Kawasaki, Kazunori; Murata, Masayuki; Ikeuchi, Yoko; Ohnishi, Shun-ichi

    1997-01-01

    The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the

  20. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses.

    PubMed

    Flipse, Jacky; Diosa-Toro, Mayra A; Hoornweg, Tabitha E; van de Pol, Denise P I; Urcuqui-Inchima, Silvio; Smit, Jolanda M

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts' antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection. PMID:27380892

  1. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses

    PubMed Central

    Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-Inchima, Silvio; Smit, Jolanda M.

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts’ antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection. PMID:27380892

  2. Fusion of Multi-Angle Imaging Spectrometer and LIDAR Data for Forest Structural Parameter Retrieval Using 3D Radiative Transfer Modeling

    NASA Astrophysics Data System (ADS)

    Rubio, J.; Sun, G.; Koetz, B.; Ranson, K. J.; Kimes, D.; Gastellu-Etchegorry, J.

    2008-12-01

    The potential of combined multi-angle/multi-spectral optical imagery and LIDAR waveform data to retrieve structural parameters on forest is explored. Our approach relies on two physically based radiative transfer models (RTM), the Discrete Anisotropic Radiative Transfer (DART) for the generation of the BRF images and Sun and Ranson's LIDAR waveform model for the large footprint LIDAR data. These RTM are based on the same basic physical principles and share common inputs parameters. We use the Zelig forest growth model to provide a synthetic but realistic data set to the two RTM. The forest canopy biophysical variables that are being investigated include the maximal tree height, fractional cover, LAI and vertical crown extension. We assess the inversion of forest structural parameters when considering each model separately, then we investigate the accuracy of a coupled inversion. Keywords: Forest, Radiative Transfer Model, Inversion, Fusion, Multi-Angle, LAI, Fractional cover, Tree height, Canopy structure, Biomass, LIDAR, Forest growth model

  3. Predicting host tropism of influenza A virus proteins using random forest

    PubMed Central

    2014-01-01

    Background Majority of influenza A viruses reside and circulate among animal populations, seldom infecting humans due to host range restriction. Yet when some avian strains do acquire the ability to overcome species barrier, they might become adapted to humans, replicating efficiently and causing diseases, leading to potential pandemic. With the huge influenza A virus reservoir in wild birds, it is a cause for concern when a new influenza strain emerges with the ability to cross host species barrier, as shown in light of the recent H7N9 outbreak in China. Several influenza proteins have been shown to be major determinants in host tropism. Further understanding and determining host tropism would be important in identifying zoonotic influenza virus strains capable of crossing species barrier and infecting humans. Results In this study, computational models for 11 influenza proteins have been constructed using the machine learning algorithm random forest for prediction of host tropism. The prediction models were trained on influenza protein sequences isolated from both avian and human samples, which were transformed into amino acid physicochemical properties feature vectors. The results were highly accurate prediction models (ACC>96.57; AUC>0.980; MCC>0.916) capable of determining host tropism of individual influenza proteins. In addition, features from all 11 proteins were used to construct a combined model to predict host tropism of influenza virus strains. This would help assess a novel influenza strain's host range capability. Conclusions From the prediction models constructed, all achieved high prediction performance, indicating clear distinctions in both avian and human proteins. When used together as a host tropism prediction system, zoonotic strains could potentially be identified based on different protein prediction results. Understanding and predicting host tropism of influenza proteins lay an important foundation for future work in constructing computation

  4. A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus

    SciTech Connect

    Zhang Shuliu; Li Li; Woodson, Sara E.; Huang, Claire Y.-H.; Kinney, Richard M.; Barrett, Alan D.T. ||||; Beasley, David W.C. |||. E-mail: d.beasley@utmb.edu

    2006-09-15

    Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.

  5. Productive infection of human immunodeficiency virus type 1 in dendritic cells requires fusion-mediated viral entry

    SciTech Connect

    Janas, Alicia M.; Dong, Chunsheng; Wang Jianhua; Wu Li

    2008-06-05

    Human immunodeficiency virus type 1 (HIV-1) enters dendritic cells (DCs) through endocytosis and viral receptor-mediated fusion. Although endocytosis-mediated HIV-1 entry can generate productive infection in certain cell types, including human monocyte-derived macrophages, productive HIV-1 infection in DCs appears to be dependent on fusion-mediated viral entry. It remains to be defined whether endocytosed HIV-1 in DCs can initiate productive infection. Using HIV-1 infection and cellular fractionation assays to measure productive viral infection and entry, here we show that HIV-1 enters monocyte-derived DCs predominately through endocytosis; however, endocytosed HIV-1 cannot initiate productive HIV-1 infection in DCs. In contrast, productive HIV-1 infection in DCs requires fusion-mediated viral entry. Together, these results provide functional evidence in understanding HIV-1 cis-infection of DCs, suggesting that different pathways of HIV-1 entry into DCs determine the outcome of viral infection.

  6. Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus.

    PubMed

    Boyington, Jeffrey C; Joyce, M Gordon; Sastry, Mallika; Stewart-Jones, Guillaume B E; Chen, Man; Kong, Wing-Pui; Ngwuta, Joan O; Thomas, Paul V; Tsybovsky, Yaroslav; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Druz, Aliaksandr; Georgiev, Ivelin S; Ko, Kiyoon; Zhou, Tongqing; Mascola, John R; Graham, Barney S; Kwong, Peter D

    2016-01-01

    Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein-stabilized in the pre-fusion (pre-F) conformation by "DS-Cav1" mutations-elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These "head-only" immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting

  7. Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus

    PubMed Central

    Boyington, Jeffrey C.; Chen, Man; Kong, Wing-Pui; Ngwuta, Joan O.; Thomas, Paul V.; Tsybovsky, Yaroslav; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Druz, Aliaksandr; Georgiev, Ivelin S.; Ko, Kiyoon; Zhou, Tongqing; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

    2016-01-01

    Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein—stabilized in the pre-fusion (pre-F) conformation by “DS-Cav1” mutations—elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These “head-only” immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity

  8. Does the major histocompatibility complex serve as a specific receptor for Semliki Forest virus?

    PubMed Central

    Oldstone, M B; Tishon, A; Dutko, F J; Kennedy, S I; Holland, J J; Lampert, P W

    1980-01-01

    Murine F9 and PCC4 teratoma cells do not express H-2 major transplantation antigens according to virus-specific T-lymphocyte cytotoxic or serological assays. However, such cells can be infected with and readily replicate many types of viruses (coxsackie B 3, mouse hepatitis, Sindbis, Semliki Forest [SFV], lymphocytic choriomeningitis, Pichinde, vesicular stomatitis, herpes simplex type 1) to the same extent as do murine F12 teratoma cells and mouse embryo fibroblasts, all of which express the H-2 determinants. In contrast, F9 and PCC4 cells are not productively infected with murine cytomegalovirus, whereas F12 and mouse embryo fibroblast cells are. In addition to replicating in H-2-negative murine teratoma cells, SFV replicates in H-2-negative murine lymphoblastoid cells. The ability of SFV to infect cells without H-2 antigens and then to effect viral antigenic expression in the cells' cytoplasm and on their surface with similar kinetics and in equivalent amounts as cells with H-2 antigens indicates that the H-2 receptor is not needed for SFV infection. Daudi cells, which lack HLA antigens, block the replication of SFV. This occurs at some point after receptor binding, as demonstrated by diminished viral mRNA. In addition, a possible membrane defect precludes viral exit in Daudi cells transfected with SFV infectious RNA. These results indicate that a cell's possession of H-2 antigens is not a requirement for SFV infection and that major histocompatibility complex antigens are not specific receptors for this virus. Images PMID:7373708

  9. Molecular evolution of the fusion protein gene in human respiratory syncytial virus subgroup A.

    PubMed

    Kimura, Hirokazu; Nagasawa, Koo; Tsukagoshi, Hiroyuki; Matsushima, Yuki; Fujita, Kiyotaka; Yoshida, Lay Myint; Tanaka, Ryota; Ishii, Haruyuki; Shimojo, Naoki; Kuroda, Makoto; Ryo, Akihide

    2016-09-01

    We studied the molecular evolution of the fusion protein (F) gene in the human respiratory syncytial virus subgroup A (HRSV-A). We performed time-scaled phylogenetic analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. We also conducted genetic distance (p-distance), positive/negative selection, and Bayesian skyline plot analyses. Furthermore, we mapped the amino acid substitutions of the protein. The MCMC-constructed tree indicated that the HRSV F gene diverged from the bovine RSV (BRSV) gene approximately 550years ago and had a relatively low substitution rate (7.59×10(-4) substitutions/site/year). Moreover, a common ancestor of HRSV-A and -B diverged approximately 280years ago, which has since formed four distinct clusters. The present HRSV-A strains were assigned six genotypes based on F gene sequences and attachment glycoprotein gene sequences. The present strains exhibited high F gene sequence similarity values and low genetic divergence. No positive selection sites were identified; however, 50 negative selection sites were identified. F protein amino acid substitutions at 17 sites were distributed in the F protein. The effective population size of the gene has remained relatively constant, but the population size of the prevalent genotype (GA2) has increased in the last 10years. These results suggest that the HRSV-AF gene has evolved independently and formed some genotypes. PMID:27291709

  10. Recombinant Newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks.

    PubMed

    Ge, Jinying; Wang, Xijun; Tian, Meijie; Gao, Yuwei; Wen, Zhiyuan; Yu, Guimei; Zhou, Weiwei; Zu, Shulong; Bu, Zhigao

    2015-05-15

    Canine Distemper Virus (CDV) infects many carnivores and cause several high-mortality disease outbreaks. The current CDV live vaccine cannot be safely used in some exotic species, such as mink and ferret. Here, we generated recombinant lentogenic Newcastle disease virus (NDV) LaSota expressing either envelope glycoproyein, heamagglutinine (H) or fusion protein (F), named as rLa-CDVH and rLa-CDVF, respectively. The feasibility of these recombinant NDVs to serve as live virus-vectored CD vaccine was evaluated in minks. rLa-CDVH induced significant neutralization antibodies (NA) to CDV and provided solid protection against virulent CDV challenge. On the contrast, rLa-CDVF induced much lower NA to CDV and fail to protected mink from virulent CDV challenge. Results suggest that recombinant NDV expressing CDV H is safe and efficient candidate vaccine against CDV in mink, and maybe other host species. PMID:25865465

  11. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    SciTech Connect

    Holzer, Georg W. . E-mail: falknef@baxter.com

    2005-07-05

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.

  12. Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma.

    PubMed

    Ketola, Anna; Hinkkanen, Ari; Yongabi, Felicitas; Furu, Petra; Määttä, Ann-Marie; Liimatainen, Timo; Pirinen, Risto; Björn, Marko; Hakkarainen, Tanja; Mäkinen, Kimmo; Wahlfors, Jarmo; Pellinen, Riikka

    2008-10-15

    Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Delta24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP-treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system. PMID:18922906

  13. The Conserved Glycine-Rich Segment Linking the N-Terminal Fusion Peptide to the Coiled Coil of Human T-Cell Leukemia Virus Type 1 Transmembrane Glycoprotein gp21 Is a Determinant of Membrane Fusion Function

    PubMed Central

    Wilson, Kirilee A.; Bär, Séverine; Maerz, Anne L.; Alizon, Marc; Poumbourios, Pantelis

    2005-01-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an ∼90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  14. The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function.

    PubMed

    Wilson, Kirilee A; Bär, Séverine; Maerz, Anne L; Alizon, Marc; Poumbourios, Pantelis

    2005-04-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an approximately 90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  15. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    SciTech Connect

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Poehlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

  16. Mapping forest biomass from space - Fusion of hyperspectral EO1-hyperion data and Tandem-X and WorldView-2 canopy height models

    NASA Astrophysics Data System (ADS)

    Kattenborn, Teja; Maack, Joachim; Faßnacht, Fabian; Enßle, Fabian; Ermert, Jörg; Koch, Barbara

    2015-03-01

    Spaceborne sensors allow for wide-scale assessments of forest ecosystems. Combining the products of multiple sensors is hypothesized to improve the estimation of forest biomass. We applied interferometric (Tandem-X) and photogrammetric (WorldView-2) based predictors, e.g. canopy height models, in combination with hyperspectral predictors (EO1-Hyperion) by using 4 different machine learning algorithms for biomass estimation in temperate forest stands near Karlsruhe, Germany. An iterative model selection procedure was used to identify the optimal combination of predictors. The most accurate model (Random Forest) reached a r2 of 0.73 with a RMSE of 14.9% (29.4 t/ha). Further results revealed that the predictive accuracy depended highly on the statistical model and the area size of the field samples. We conclude that a fusion of canopy height and spectral information allows for accurate estimations of forest biomass from space.

  17. Functional Properties and Genetic Relatedness of the Fusion and Hemagglutinin-Neuraminidase Proteins of a Mumps Virus-Like Bat Virus

    PubMed Central

    Krüger, Nadine; Hoffmann, Markus; Drexler, Jan Felix; Müller, Marcel Alexander; Corman, Victor Max; Sauder, Christian; Rubin, Steven; He, Biao; Örvell, Claes; Drosten, Christian

    2015-01-01

    ABSTRACT A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of

  18. The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components

    PubMed Central

    Laliberte, Jason P.; Weisberg, Andrea S.; Moss, Bernard

    2011-01-01

    For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry. PMID:22194690

  19. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

    PubMed

    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

  20. Analysis of the selective advantage conferred by a C-E1 fusion protein synthesized by rubella virus DI RNAs

    SciTech Connect

    Claus, Claudia; Tzeng, W.-P.; Liebert, Uwe Gerd; Frey, Teryl K.

    2007-12-05

    During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and

  1. LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation.

    PubMed

    Kim, Shin-Hee; Xiao, Sa; Collins, Peter L; Samal, Siba K

    2016-06-01

    The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors. PMID:26932300

  2. Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

    PubMed Central

    Samal, Sweety; Khattar, Sunil K.; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L.

    2013-01-01

    The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. PMID:23843643

  3. Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Dragic, T; Picard, L; Alizon, M

    1995-01-01

    Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry. PMID:7815477

  4. New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

    PubMed Central

    Takahashi, Yuto; Tomotsune, Daihachiro; Takizawa, Sakiko; Yue, Fengming; Nagai, Mika; Yokoyama, Tadayuki; Hirashima, Kanji; Sasaki, Katsunori

    2015-01-01

    AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. METHODS: Cardiomyocytes (1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. CONCLUSION: The present results show that close contacts were acquired and facilitated

  5. Mutational Analysis of the Candidate Internal Fusion Peptide of the Avian Leukosis and Sarcoma Virus Subgroup A Envelope Glycoprotein

    PubMed Central

    Hernandez, Lorraine D.; White, Judith M.

    1998-01-01

    The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Val30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide. PMID:9525653

  6. The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

    SciTech Connect

    Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo . E-mail: Riccardo.Wittek@unil.ch

    2005-07-05

    The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F{sub WT}) and attachment (H{sub WT}) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H{sub WT} determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F{sub WT} reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

  7. Evidence that maturation of the N-linked glycans of the respiratory syncytial virus (RSV) glycoproteins is required for virus-mediated cell fusion: The effect of {alpha}-mannosidase inhibitors on RSV infectivity

    SciTech Connect

    McDonald, Terence P.; Jeffree, Chris E.; Li, Ping; Rixon, Helen W. McL.; Brown, Gaie; Aitken, James D.; MacLellan, Kirsty; Sugrue, Richard J. . E-mail: rjsugrue@ntu.edu.sg

    2006-07-05

    Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the {alpha}-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affected by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.

  8. Revealing the binding mode between respiratory syncytial virus fusion protein and benzimidazole-based inhibitors.

    PubMed

    Ji, Dingjue; Ye, Wei; Chen, HaiFeng

    2015-07-01

    Human respiratory syncytial virus (HRSV) is a major respiratory pathogen in newborn infants and young children and can also be a threat to some elderly and high-risk adults with chronic pulmonary disease and the severely immunocompromised. The RSV fusion (RSVF) protein has been an attractive target for vaccine and drug development. Experimental results indicate a series of benzimidazole-based inhibitors which target RSVF protein to inhibit the viral entry of RSV. To reveal the binding mode between these inhibitors and RSVF protein, molecular docking and molecular dynamics simulations were used to investigate the interactions between the inhibitors and the core domain of RSVF protein. MD results suggest that the active molecules have stronger π-π stacking, cation-π, and other interactions than less active inhibitors. The binding free energy between the active inhibitor and RSVF protein is also found to be significantly lower than that of the less active one using MM/GBSA. Then, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) methods were used to construct three dimensional quantitative structure-activity (3D-QSAR) models. The cross-validated q(2) values are found to be 0.821 and 0.795 for CoMFA and CoMSIA, respectively. And the non-cross-validated r(2) values are 0.973 and 0.961. Ninety-two test set compounds validated these models. The results suggest that these models are robust with good prediction abilities. Furthermore, these models reveal possible methods to improve the bioactivity of inhibitors. PMID:25872614

  9. A single amino acid in the F2 subunit of respiratory syncytial virus fusion protein alters growth and fusogenicity

    PubMed Central

    Schickli, Jeanne H.; Tang, Roderick S.

    2013-01-01

    Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children, especially in infants less than 1 year of age. There are currently no licensed vaccines against RSV. rA2ΔM2-2 is a promising live-attenuated vaccine candidate that is currently being evaluated in the clinic. Attenuation of rA2ΔM2-2 is achieved by a single deletion of the M2-2 gene, which disrupts the balance between viral transcription and replication. Whilst performing a manufacturing feasibility study in a serum-free adapted Vero cell line, differences in growth kinetics and cytopathic effect (CPE) were identified between two rA2ΔM2-2 vaccine candidates. Comparative sequence analysis identified four amino acid differences between the two vaccine viruses. Recombinant rA2ΔM2-2 viruses carrying each of the four amino acid differences identified a K66E mutation in the F2 fragment of the fusion (F) protein as the cause of the growth and CPE differences. Syncytium-formation experiments with RSV F protein carrying mutations at aa 66 suggested that a change in charge at this residue within the F2 fragment can have a significant impact on fusion. PMID:24092758

  10. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    SciTech Connect

    Maric, Martina; Haugo, Alison C.; Dauer, William; Johnson, David; Roller, Richard J.

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  11. Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

    PubMed

    Rothan, Hussin A; Bahrani, Hirbod; Mohamed, Zulqarnain; Abd Rahman, Noorsaadah; Yusof, Rohana

    2014-01-01

    Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities. PMID:24722532

  12. A yellow fever epizootic in Zika forest, Uganda, during 1972: Part 1: Virus isolation and sentinel monkeys.

    PubMed

    Kirya, B G

    1977-01-01

    The results of the yellow fever immunity survey of Central and East Africa reported by SAWYER & WHITMAN in 1936 prompted scientists to undertake well-planned epidemiological studies on yellow fever in eastern Africa. A Yellow Fever Research Institute (the present East African Virus Research Institute) was established at Entebbe in 1936 for this purpose. One of the areas where much work has been carried out is a strip of typical tropical forest, the Zika Forest, 12 kilometres from the Institute. Routine surveillance work, particularly on the biting activity of the yellow fever vector mosquitoes, has been going on since 1946. It was during one of these studies in 1972 that the first yellow fever virus strain was isolated from Aedes africanus collected from the Zika and Sisa forests and one strain was isolated from Coquillettidia fuscopennata, also from the Zika Forest. Three sentinel rhesus monkeys, nomimmune to YF, which were kept in the Zika Forest during the time of the epizootic died of YF disease. The present observations indicate that YF is still present in Africa, and as such it still remains a potential menace to the human population. The epidemiological implications are discussed. PMID:407675

  13. Influenza virus vaccine expressing fusion and attachment protein epitopes of respiratory syncytial virus induces protective antibodies in BALB/c mice.

    PubMed

    Bian, Chengrong; Liu, Shuzhen; Liu, Na; Zhang, Guangzhou; Xing, Li; Song, Yingwei; Duan, Yueqiang; Gu, Hongjing; Zhou, Ya; Zhang, Peirui; Li, Zhiwei; Zhang, Keming; Wang, Zhaohai; Zhang, Shaogeng; Wang, Xiliang; Yang, Penghui

    2014-04-01

    Respiratory syncytial virus (RSV) is an important viral pathogen that causes life-threatening respiratory infections in both infants and the elderly; no vaccines are at present available. In this report, we examined the use of influenza virus as a vehicle for production of an experimental RSV vaccine. We used reverse genetics to generate a recombinant influenza A virus with epitopes from the RSV fusion (F) and attachment (G) proteins (rFlu/RSV/F+G) in the influenza virus nonstructural (NS1) protein gene. Expression of RSV F+G epitope proteins was confirmed by Western blotting, and no changes in viral morphology were evident following examination by electron microscopy. BALB/c mice immunized intranasally with rFlu/RSV/F+G showed viral-specific antibody responses against both influenza and RSV. Total IgG, IgG1, IgG2a and IgA were measured in mice immunized with rFlu/RSV/F+G, revealing robust cellular and mucosal immune responses. Furthermore, we found that rFlu/RSV/F+G conferred protection against subsequent influenza and RSV challenges, showing significant decreases in viral replication and obvious attenuation of histopathological changes associated with viral infections. These findings suggest that rFlu/RSV/F+G is a promising vaccine candidate, which should be further assessed using cotton rat and primate models. PMID:24509239

  14. Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

    SciTech Connect

    Zhang, Jufeng; Wang, Zhanli; Wei, Fang; Qiu, Wei; Zhang, Liangren; Huang, Qian . E-mail: qhuang@sjtu.edu.cn

    2007-08-17

    Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.

  15. Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

    SciTech Connect

    Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.; Erb, Steven M.; Luy, Betty E.; Calvert, Amanda E.; Blair, Carol D.; Roehrig, John T.; Huang, Claire Y.-H.

    2011-04-25

    Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.

  16. A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

    PubMed

    Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

    2016-01-01

    Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model. PMID:27612283

  17. Analysis of the Selective Advantage Conferred by a C-E1 Fusion Protein Synthesized by Rubella Virus DI RNAs

    PubMed Central

    Claus, Claudia; Tzeng, Wen-Pin; Liebert, Uwe Gerd; Frey, Teryl K.

    2009-01-01

    During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for maintenance of the DI (Tzeng & Frey, 2006). A BHK cell line stably expressing the RUB structural proteins was established which was used to package DI’s into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from two to four days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and sustaining infected cell percentage increases the likelihood of co-infection by a DI and wt RUB during serial passage thus

  18. Forest Attributes from Radar Interferometric Structure and its Fusion with Optical Remote Sensing

    NASA Technical Reports Server (NTRS)

    Treuhaft, Robert N.; Law, Beverly E.; Asner, Gregory P.

    2004-01-01

    The possibility of global, three-dimensional remote sensing of forest structure with interferometric synthetic aperture radar (InSAR) bears on important forest ecological processes, particularly the carbon cycle. InSAR supplements two-dimensional remote sensing with information in the vertical dimension. Its strengths in potential for global coverage complement those of lidar (light detecting and ranging), which has the potential for high-accuracy vertical profiles over small areas. InSAR derives its sensitivity to forest vertical structure from the differences in signals received by two, spatially separate radar receivers. Estimation of parameters describing vertical structure requires multiple-polarization, multiple-frequency, or multiple-baseline InSAR. Combining InSAR with complementary remote sensing techniques, such as hyperspectral optical imaging and lidar, can enhance vertical-structure estimates and consequent biophysical quantities of importance to ecologists, such as biomass. Future InSAR experiments will supplement recent airborne and spaceborne demonstrations, and together with inputs from ecologists regarding structure, they will suggest designs for future spaceborne strategies for measuring global vegetation structure.

  19. Induction of Heterosubtypic Cross-Protection against Influenza by a Whole Inactivated Virus Vaccine: The Role of Viral Membrane Fusion Activity

    PubMed Central

    Budimir, Natalija; Huckriede, Anke; Meijerhof, Tjarko; Boon, Louis; Gostick, Emma; Price, David A.; Wilschut, Jan; de Haan, Aalzen

    2012-01-01

    Background The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored. Methodology/Principal Findings In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge. Conclusion/Significance The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity

  20. The Roles of Histidines and Charged Residues as Potential Triggers of a Conformational Change in the Fusion Loop of Ebola Virus Glycoprotein

    PubMed Central

    Lee, Jinwoo; Gregory, Sonia M.; Nelson, Elizabeth A.; White, Judith M.; Tamm, Lukas K.

    2016-01-01

    Ebola virus (EBOV) enters cells from late endosomes/lysosomes under mildly acidic conditions. Entry by fusion with the endosomal membrane requires the fusion loop (FL, residues 507–560) of the EBOV surface glycoprotein to undergo a pH-dependent conformational change. To find the pH trigger for this reaction we mutated multiple conserved histidines and charged and uncharged hydrophilic residues in the FL and measured their activity by liposome fusion and cell entry of virus-like particles. The FL location in the membrane was assessed by NMR using soluble and lipid-bound paramagnetic relaxation agents. While we could not identify a single residue to be alone responsible for pH triggering, we propose that a distributed pH effect over multiple residues induces the conformational change that enhances membrane insertion and triggers the fusion activity of the EBOV FL. PMID:27023721

  1. Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

    SciTech Connect

    Ruel, Nancy . E-mail: n-ruel@northwestern.edu; Zago, Anna . E-mail: anna_zago@acgtinc.com; Spear, Patricia G. . E-mail: p-spear@northwestern.edu

    2006-03-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.

  2. Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

    PubMed

    Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

    2011-09-01

    Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC. PMID:21361787

  3. Limited Effects of Type I Interferons on Kyasanur Forest Disease Virus in Cell Culture

    PubMed Central

    Cook, Bradley W. M.; Ranadheera, Charlene; Nikiforuk, Aidan M.; Cutts, Todd A.; Kobasa, Darwyn; Court, Deborah A.; Theriault, Steven S.

    2016-01-01

    Background The tick-borne flavivirus, Kyasanur Forest disease virus (KFDV) causes seasonal infections and periodic outbreaks in south-west India. The current vaccine offers poor protection with reported issues of coverage and immunogenicity. Since there are no approved prophylactic therapeutics for KFDV, type I IFN-α/β subtypes were assessed for antiviral potency against KFDV in cell culture. Methodology/Principal Findings The continued passage of KFDV-infected cells with re-administered IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production whereas the IFN-sensitive, green fluorescent protein-expressing vesicular stomatitis virus (VSV-GFP) infection was controlled. Further evaluation of the other IFN-α/β subtypes versus KFDV infection indicated that single treatments of either IFN-αWA and IFN-αΚ appeared to be more effective than IFN-α2a at reducing KFDV titres. Concentration-dependent analysis of these IFN-α/β subtypes revealed that regardless of subtype, low concentrations of IFN were able to limit cytopathic effects (CPE), while significantly higher concentrations were needed for inhibition of virion release. Furthermore, expression of the KFDV NS5 in cell culture before IFN addition enabled VSV-GFP to overcome the effects of IFN-α/β signalling, producing a robust infection. Conclusions/Significance Treatment of cell culture with IFN does not appear to be suitable for KFDV eradication and the assay used for such studies should be carefully considered. Further, it appears that the NS5 protein is sufficient to permit KFDV to bypass the antiviral properties of IFN. We suggest that other prophylactic therapeutics should be evaluated in place of IFN for treatment of individuals with KFDV disease. PMID:27479197

  4. Surveillance should be strengthened to improve epidemiological understandings of mosquito-borne Barmah Forest virus infection

    PubMed Central

    Eastwood, Keith; Webb, Cameron; Durrheim, David

    2012-01-01

    Introduction Barmah Forest virus (BFV) is a mosquito-borne virus causing epidemic polyarthritis in Australia. This study used case follow-up of cases from the surveillance system to demonstrate that routinely collected BFV notification data were an unreliable indicator of the true location of exposure. Methods BFV notifications from June 2001 to May 2011 were extracted from the New South Wales (NSW) Notifiable Conditions Information Management System to study case distribution. Disease cluster analysis was performed using spatial scan statistics. Exposure history data were collected from cases notified in 2010 and 2011 to accurately determine travel to high-risk areas. Results Cluster analysis using address data identified an area of increased BFV disease incidence in the mid-north coast of NSW contiguous with estuarine wetlands. When travel to this area was investigated, 96.7% (29/30) cases reported having visited coastal regions within four weeks of developing symptoms. Discussion Along the central NSW coastline, extensive wetlands occur in close proximity to populated areas. These wetlands provide ideal breeding habitats for a range of mosquito species implicated in the transmission of BFV. This is the first study to fully assess case exposure with findings suggesting that sporadic cases of BFV in people living further away from the coast do not reflect alternative exposure sites but are likely to result from travel to coastal regions. Spatial analysis by case address alone may lead to inaccurate understandings of the true distribution of arboviral diseases. Subsequently, this information has important implications for the collection of mosquito-borne disease surveillance information and public health response strategies. PMID:23908926

  5. pH-Dependent Vesicle Fusion Induced by the Ectodomain of the Human Immunodeficiency Virus Membrane Fusion Protein gp41: Two Kinetically Distinct Processes and Fully-Membrane-Associated gp41 with Predominant β Sheet Fusion Peptide Conformation

    PubMed Central

    Ratnayake, Punsisi U.; Sackett, Kelly; Nethercott, Matthew J.; Weliky, David P.

    2014-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The FP is critical for fusion and is hypothesized to bind to the host cell membrane. Large ectodomain constructs either with or without the FP are highly aggregated at physiologic pH but soluble in the pH 3–4 range with hyperthermostable hairpin structure with antiparallel NHR and CHR helices. The present study focuses on the large gp41 ectodomain constructs “Hairpin” (HP) containing NHR+loop+CHR and “FP-Hairpin” (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH 4 where the protein is positively-charged but do not induce fusion at pH 7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. The functional role of the hydrophobic FP is supported by increases in the rate and extent of fusion for FP-HP relative to HP. There are two kinetically distinct fusion processes at pH 4: (1) a faster ~100 ms−1 process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms−1 process with extent strongly inversely correlated with this charge. The faster charge-dependent process is likely related to the electrostatic energy released upon initial monomer protein binding to the vesicle. After dissipation of this energy, the subsequent slower process is likely due to the equilibrium membrane-associated structure of the protein. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region

  6. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    SciTech Connect

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  7. Immunogenicity and protective efficacy of recombinant fusion proteins containing spike protein of infectious bronchitis virus and hemagglutinin of H3N2 influenza virus in chickens.

    PubMed

    Yin, Lijuan; Zeng, Yuyao; Wang, Wei; Wei, Ying; Xue, Chunyi; Cao, Yongchang

    2016-09-01

    Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens and vaccination is the main method for disease control. The S1 protein, which contains several virus neutralization epitopes, is considered to be a target site of vaccine development. However, although protective immune responses could be induced by recombinant S1 protein, the protection rate in chickens was still low (<50%). Here, we generated fused S1 proteins with HA2 protein (rS1-HA2) or transmembrane domain and cytoplasmic tail (rS1-H3(TM)) from hemagglutinin of H3N2 influenza virus. After immunization, animals vaccinated with fusion proteins rS1-HA2 and rS1-H3(TM) demonstrated stronger robust humoral and cellular immune responses than that of rS1 and inactivated M41 vaccine. The protection rates of groups immunized with rS1-HA2 (87%) were significantly higher than the groups inoculated with rS1 (47%) and inactivated M41 vaccine (53%). And chickens injected with rS1-H3(TM) had similar level of protection (73%) comparing to chickens vaccinated with rS1 (47%) (P=0.07). Our data suggest that S1 protein fused to the HA2 or TM proteins from hemagglutinin of H3N2 influenza virus may provide a new strategy for high efficacy recombinant vaccine development against IBV. PMID:27497621

  8. Myxoma Virus Expressing a Fusion Protein of Interleukin-15 (IL15) and IL15 Receptor Alpha Has Enhanced Antitumor Activity

    PubMed Central

    Tosic, Vesna; Thomas, Diana L.; Kranz, David M.; Liu, Jia; McFadden, Grant; Shisler, Joanna L.; MacNeill, Amy L.; Roy, Edward J.

    2014-01-01

    Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8+ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system. PMID:25329832

  9. Mapping the distribution of maize streak virus genotypes across the forest and transition zones of Ghana.

    PubMed

    Oppong, Allen; Offei, Samuel K; Ofori, Kwadwo; Adu-Dapaah, Hans; Lamptey, Joseph N L; Kurenbach, Brigitta; Walters, Matthew; Shepherd, Dionne N; Martin, Darren P; Varsani, Arvind

    2015-02-01

    Throughout sub-Saharan Africa, maize streak virus strain A (MSV-A), the causal agent of maize streak disease (MSD), is an important biological constraint on maize production. In November/December 2010, an MSD survey was carried out in the forest and transition zones of Ghana in order to obtain MSV-A virulence sources for the development of MSD-resistant maize genotypes with agronomic properties suitable for these regions. In 79 well-distributed maize fields, the mean MSD incidence was 18.544 % and the symptom severity score was 2.956 (1 = no symptoms and 5 = extremely severe). We detected no correlation between these two variables. Phylogenetic analysis of cloned MSV-A isolates that were fully sequenced from samples collected in 51 of these fields, together with those sampled from various other parts of Africa, indicated that all of the Ghanaian isolates occurred within a broader cluster of West African isolates, all belonging to the highly virulent MSV-A1 subtype. Besides being the first report of a systematic MSV survey in Ghana, this study is the first to characterize the full-genome sequences of Ghanaian MSV isolates. The 51 genome sequences determined here will additionally be a valuable resource for the rational selection of representative MSV-A variant panels for MSD resistance screening. PMID:25344899

  10. Weather Variability, Tides, and Barmah Forest Virus Disease in the Gladstone Region, Australia

    PubMed Central

    Naish, Suchithra; Hu, Wenbiao; Nicholls, Neville; Mackenzie, John S.; McMichael, Anthony J.; Dale, Pat; Tong, Shilu

    2006-01-01

    In this study we examined the impact of weather variability and tides on the transmission of Barmah Forest virus (BFV) disease and developed a weather-based forecasting model for BFV disease in the Gladstone region, Australia. We used seasonal autoregressive integrated moving-average (SARIMA) models to determine the contribution of weather variables to BFV transmission after the time-series data of response and explanatory variables were made stationary through seasonal differencing. We obtained data on the monthly counts of BFV cases, weather variables (e.g., mean minimum and maximum temperature, total rainfall, and mean relative humidity), high and low tides, and the population size in the Gladstone region between January 1992 and December 2001 from the Queensland Department of Health, Australian Bureau of Meteorology, Queensland Department of Transport, and Australian Bureau of Statistics, respectively. The SARIMA model shows that the 5-month moving average of minimum temperature (β = 0.15, p-value < 0.001) was statistically significantly and positively associated with BFV disease, whereas high tide in the current month (β = −1.03, p-value = 0.04) was statistically significantly and inversely associated with it. However, no significant association was found for other variables. These results may be applied to forecast the occurrence of BFV disease and to use public health resources in BFV control and prevention. PMID:16675420

  11. Herpes Simplex Virus 1 gN Partners with gM To Modulate the Viral Fusion Machinery

    PubMed Central

    El Kasmi, Imane

    2014-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes, but their roles in viral nuclear egress are unclear. Among them, glycoprotein M (gM), a known modulator of virus-induced membrane fusion, is distributed on both the inner and outer nuclear membranes at the early stages of the infection, when no other viral glycoproteins are yet present there. Later on, it is found on perinuclear virions and ultimately redirected to the trans-Golgi network (TGN), where it cycles with the cell surface. In contrast, transfected gM is found only at the TGN and cell surface, hinting at an interaction with other viral proteins. Interestingly, many herpesvirus gM analogs interact with their gN counterparts, which typically alters their intracellular localization. To better understand how HSV-1 gM localization is regulated, we evaluated its ability to bind gN and discovered it does so in both transfected and infected cells, an interaction strongly weakened by the deletion of the gM amino terminus. Functionally, while gN had no impact on gM localization, gM redirected gN from the endoplasmic reticulum (ER) to the TGN. Most interestingly, gN overexpression stimulated the formation of syncytia in the context of an infection by a nonsyncytial strain, indicating that gM and gN not only physically but also functionally interact and that gN modulates gM's activity on membrane fusion. IMPORTANCE HSV-1 gM is an important modulator of virally induced cell-cell fusion and viral entry, a process that is likely finely modulated in time and space. Until now, little was known of the proteins that regulate gM's activity. In parallel, gM is found in various intracellular locations at different moments, ranging from nuclear membranes, perinuclear virions, the TGN, cell

  12. Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head.

    PubMed

    Avila, Mislay; Khosravi, Mojtaba; Alves, Lisa; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Plemper, Richard K; Plattet, Philippe

    2015-01-15

    Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F's interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways. PMID:25355896

  13. Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor*

    PubMed Central

    Cook, Jonathan D.; Soto-Montoya, Hazel; Korpela, Markus K.; Lee, Jeffrey E.

    2015-01-01

    Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion. PMID:26082488

  14. Fusion of AIRSAR and TM Data for Parameter Classification and Estimation in Dense and Hilly Forests

    NASA Technical Reports Server (NTRS)

    Moghaddam, Mahta; Dungan, J. L.; Coughlan, J. C.

    2000-01-01

    The expanded remotely sensed data space consisting of coincident radar backscatter and optical reflectance data provides for a more complete description of the Earth surface. This is especially useful where many parameters are needed to describe a certain scene, such as in the presence of dense and complex-structured vegetation or where there is considerable underlying topography. The goal of this paper is to use a combination of radar and optical data to develop a methodology for parameter classification for dense and hilly forests, and further, class-specific parameter estimation. The area to be used in this study is the H. J. Andrews Forest in Oregon, one of the Long-Term Ecological Research (LTER) sites in the US. This area consists of various dense old-growth conifer stands, and contains significant topographic relief. The Andrews forest has been the subject of many ecological studies over several decades, resulting in an abundance of ground measurements. Recently, biomass and leaf-area index (LAI) values for approximately 30 reference stands have also become available which span a large range of those parameters. The remote sensing data types to be used are the C-, L-, and P-band polarimetric radar data from the JPL airborne SAR (AIRSAR), the C-band single-polarization data from the JPL topographic SAR (TOPSAR), and the Thematic Mapper (TM) data from Landsat, all acquired in late April 1998. The total number of useful independent data channels from the AIRSAR is 15 (three frequencies, each with three unique polarizations and amplitude and phase of the like-polarized correlation), from the TOPSAR is 2 (amplitude and phase of the interferometric correlation), and from the TM is 6 (the thermal band is not used). The range pixel spacing of the AIRSAR is 3.3m for C- and L-bands and 6.6m for P-band. The TOPSAR pixel spacing is 10m, and the TM pixel size is 30m. To achieve parameter classification, first a number of parameters are defined which are of interest to

  15. Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease

    PubMed Central

    Fourrier, Mickael; Lester, Katherine; Markussen, Turhan; Falk, Knut; Secombes, Christopher J.; McBeath, Alastair; Collet, Bertrand

    2015-01-01

    In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes. PMID:26517828

  16. Enhanced expression of rabies virus surface G-protein in Escherichia coli using SUMO fusion.

    PubMed

    Singh, Ankit; Yadav, Dinesh; Rai, Krishan Mohan; Srivastava, Meenal; Verma, Praveen C; Singh, Pradhyumna K; Tuli, Rakesh

    2012-01-01

    Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research. PMID:22134654

  17. pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

    PubMed Central

    Shilova, L. A.; Kachala, M. V.; Tashkin, V. Y.; Sokolov, V. S.; Fedorova, N. V.; Baratova, L. A.; Knyazev, D. G.; Zimmerberg, J.; Chizmadzhev, Y. A.

    2015-01-01

    ABSTRACT Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ∼6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ∼5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. IMPORTANCE Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to

  18. Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

    SciTech Connect

    Oh, Sang Kon . E-mail: sangkono@baylorhealth.edu; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A. . E-mail: berzofsk@helix.nih.gov

    2006-09-01

    Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8{sup +} T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8{sup +} T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K{sup b} and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection.

  19. Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes

    PubMed Central

    Firouzamandi, Masoumeh; Moeini, Hassan; Hosseini, Davood; Bejo, Mohd Hair; Omar, Abdul Rahman; Mehrbod, Parvaneh

    2016-01-01

    The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure. PMID:27051336

  20. Nucleotide sequence of a region of the herpes simplex virus type 1 gB glycoprotein gene: mutations affecting rate of virus entry and cell fusion.

    PubMed

    Bzik, D J; Fox, B A; DeLuca, N A; Person, S

    1984-08-01

    The tsB5 isolate of herpes simplex virus type I (HSV-1) enters host cells more rapidly than does KOS, an independent isolate of HSV-1, and this rate-of-entry determinant is located between prototypic map coordinates 0.350 and 0.360 (1). The nucleotide sequence of strain tsB5 has now been determined between prototypic map coordinates 0.347 and 0.360. Comparison of the tsB5 sequence to the homologous KOS sequence revealed that the rate-of-entry difference between these two HSV-1 strains may be due to the single amino acid difference observed within these sequences (0.350 to 0.360). A cell fusion determinant in tsB5 is located between coordinates 0.345 and 0.355 and to the left of the rate-of-entry determinant (1). Nucleotide sequence analysis revealed a second amino acid difference between tsB5 and KOS at coordinate 0.349. The cell fusion determinant was tentatively assigned to this location. PMID:6089415

  1. Protection against lethal measles virus infection in mice by immune-stimulating complexes containing the hemagglutinin or fusion protein.

    PubMed Central

    Varsanyi, T M; Morein, B; Löve, A; Norrby, E

    1987-01-01

    The importance of each of the two surface glycoproteins of measles virus in active and passive immunization was examined in mice. Infected-cell lysates were depleted of either the hemagglutinin (H) or fusion (F) glycoprotein by using multiple cycles of immunoaffinity chromatography. The products were used to prepare immune-stimulating complexes (iscoms) containing either F or H glycoprotein. Such complexes are highly immunogenic, possibly as a result of effective presentation of viral proteins to the immune system [B. Morein, B. Sundquist, S. Höglund, K. Dalsgaard, and A. Osterhaus, Nature (London) 308:457-460, 1984]. Groups of 3-week-old BALB/c mice were inoculated with the iscom preparations. All animals developed hemolysis-inhibiting antibodies, whereas only sera of animals immunized with the iscoms containing the H glycoprotein had hemagglutination-inhibiting antibodies. Sera from animals immunized with the H or F preparation only precipitated the homologous glycoprotein in radioimmune precipitation assays. The immunized animals were challenged with a lethal dose of the hamster neurotropic variant of measles virus. Of the 7-week-old animals in the nonimmunized control group, 50% died within 10 days after challenge. No animals in the immunized groups showed symptoms of disease throughout the observation period of 3 months. Passive administration of anti-H monoclonal antibodies gave full protection against the 100% lethal acute infection with the hamster neurotropic variant of measles virus in newborn mice, whereas anti-F monoclonal antibodies failed to protect the animals. This study emphasizes that both H and F glycoproteins need to be considered in the development of measles virus subunit vaccines. Images PMID:2960833

  2. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

    PubMed Central

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F.

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  3. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    PubMed

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  4. Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

    PubMed

    Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

    2015-11-15

    Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. PMID:26022509

  5. Lister strain of vaccinia virus armed with endostatin-angiostatin fusion gene as a novel therapeutic agent for human pancreatic cancer.

    PubMed

    Tysome, J R; Briat, A; Alusi, G; Cao, F; Gao, D; Yu, J; Wang, P; Yang, S; Dong, Z; Wang, S; Deng, L; Francis, J; Timiryasova, T; Fodor, I; Lemoine, N R; Wang, Y

    2009-10-01

    Survival after pancreatic cancer remains poor despite incremental advances in surgical and adjuvant therapy, and new strategies for treatment are needed. Oncolytic virotherapy is an attractive approach for cancer treatment. In this study, we have evaluated the effectiveness of the Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapeutic approach for pancreatic cancer. The Lister vaccine strain of vaccinia virus was effective against all human pancreatic carcinoma cells tested in vitro, especially those insensitive to oncolytic adenovirus. The virus displayed inherently high selectivity for cancer cells, sparing normal cells both in vitro and in vivo, with effective infection of tumors after both intravenous (i.v.) and intratumoral (i.t.) administrations. The expression of the endostatin-angiostatin fusion protein was confirmed in a pancreatic cancer model both in vitro and in vivo, with evidence of inhibition of angiogenesis. This novel vaccinia virus showed significant antitumor potency in vivo against the Suit-2 model by i.t. administration. This study suggests that the novel Lister strain of vaccinia virus armed with the endostatin-angiostatin fusion gene is a potential therapeutic agent for pancreatic cancer. PMID:19587709

  6. Spatio-Temporal Patterns of Barmah Forest Virus Disease in Queensland, Australia

    PubMed Central

    Naish, Suchithra; Hu, Wenbiao; Mengersen, Kerrie; Tong, Shilu

    2011-01-01

    Background Barmah Forest virus (BFV) disease is a common and wide-spread mosquito-borne disease in Australia. This study investigated the spatio-temporal patterns of BFV disease in Queensland, Australia using geographical information system (GIS) tools and geostatistical analysis. Methods/Principal Findings We calculated the incidence rates and standardised incidence rates of BFV disease. Moran's I statistic was used to assess the spatial autocorrelation of BFV incidences. Spatial dynamics of BFV disease was examined using semi-variogram analysis. Interpolation techniques were applied to visualise and display the spatial distribution of BFV disease in statistical local areas (SLAs) throughout Queensland. Mapping of BFV disease by SLAs reveals the presence of substantial spatio-temporal variation over time. Statistically significant differences in BFV incidence rates were identified among age groups (χ2 = 7587, df = 7327,p<0.01). There was a significant positive spatial autocorrelation of BFV incidence for all four periods, with the Moran's I statistic ranging from 0.1506 to 0.2901 (p<0.01). Semi-variogram analysis and smoothed maps created from interpolation techniques indicate that the pattern of spatial autocorrelation was not homogeneous across the state. Conclusions/Significance This is the first study to examine spatial and temporal variation in the incidence rates of BFV disease across Queensland using GIS and geostatistics. The BFV transmission varied with age and gender, which may be due to exposure rates or behavioural risk factors. There are differences in the spatio-temporal patterns of BFV disease which may be related to local socio-ecological and environmental factors. These research findings may have implications in the BFV disease control and prevention programs in Queensland. PMID:22022430

  7. A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains.

    PubMed Central

    Berson, J F; Long, D; Doranz, B J; Rucker, J; Jirik, F R; Doms, R W

    1996-01-01

    Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains. PMID:8709256

  8. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease.

    PubMed

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin; Lee, Jong Seok; Kim, Yu-Jin; Lee, Yu-Na; Kim, Min-Chul; Cho, Minkyoung; Kang, Sang-Moo

    2016-07-01

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. PMID:27123586

  9. Full Inactivation of Human Influenza Virus by High Hydrostatic Pressure Preserves Virus Structure and Membrane Fusion While Conferring Protection to Mice against Infection

    PubMed Central

    Dumard, Carlos H.; Barroso, Shana P. C.; de Oliveira, Guilherme A. P.; Carvalho, Carlos A. M.; Gomes, Andre M. O.; Couceiro, José Nelson S. S.; Ferreira, Davis F.; Nico, Dirlei; Oliveira, Andrea C.; Silva, Jerson L.; Santos, Patrícia S.

    2013-01-01

    Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses. PMID:24282553

  10. [Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yang, Yang; Wen, Bo; Tan, Wenjie

    2015-01-01

    To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine. PMID:25997323

  11. Fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein.

    PubMed Central

    Paterson, R G; Harris, T J; Lamb, R A

    1984-01-01

    The nucleotide sequence of the mRNA coding for the fusion glycoprotein (F) of the paramyxovirus, simian virus 5, has been obtained. There is a single large open reading frame on the mRNA that encodes a protein of 529 amino acids with a molecular weight of 56,531. The proteolytic cleavage/activation site of F, to yield F2 and F1, contains five arginine residues. Six potential glycosylation sites were identified in the protein, two on F2 and four on F1. The deduced amino acid sequence indicates that F is extensively hydrophobic over the length of the polypeptide chain. Three regions are very hydrophobic and could interact directly with membranes: these are the NH2-terminal putative signal peptide, the COOH-terminal putative membrane anchorage domain, and the NH2-terminal region of F1. Images PMID:6093114

  12. Parainfluenza virus type 3 expressing the native or soluble fusion (F) Protein of Respiratory Syncytial Virus (RSV) confers protection from RSV infection in African green monkeys.

    PubMed

    Tang, Roderick S; MacPhail, Mia; Schickli, Jeanne H; Kaur, Jasmine; Robinson, Christopher L; Lawlor, Heather A; Guzzetta, Jeanne M; Spaete, Richard R; Haller, Aurelia A

    2004-10-01

    Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines. PMID:15452239

  13. Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

    SciTech Connect

    Bellamy-McIntyre, Anna K.; Baer, Severine; Ludlow, Louise; Drummer, Heidi E.; Poumbourios, Pantelis

    2010-04-16

    The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1{sub QH1549.13} blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.

  14. Interaction between the G3 and L5 proteins of the vaccinia virus entry-fusion complex

    SciTech Connect

    Wolfe, Cindy L.; Moss, Bernard

    2011-04-10

    The vaccinia virus entry-fusion complex (EFC) consists of 10 to 12 proteins that are embedded in the viral membrane and individually required for fusion with the cell and entry of the core into the cytoplasm. The architecture of the EFC is unknown except for information regarding two pair-wise interactions: A28 with H2 and A16 with G9. Here we used a technique to destabilize the EFC by repressing the expression of individual components and identified a third pair-wise interaction: G3 with L5. These two proteins remained associated under several different EFC destabilization conditions and in each case were immunopurified together as demonstrated by Western blotting. Further evidence for the specific interaction of G3 and L5 was obtained by mass spectrometry. This interaction also occurred when G3 and L5 were expressed in uninfected cells, indicating that no other viral proteins were required. Thus, the present study extends our knowledge of the protein interactions important for EFC assembly and stability.

  15. Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

    PubMed Central

    Atanasiu, Doina; Whitbeck, J. Charles; Cairns, Tina M.; Reilly, Brigid; Cohen, Gary H.; Eisenberg, Roselyn J.

    2007-01-01

    Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell–cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell–cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry. PMID:18003913

  16. Enzootic Arbovirus Surveillance in Forest Habitat and Phylogenetic Characterization of Novel Isolates of Gamboa Virus in Panama.

    PubMed

    Eastwood, Gillian; Loaiza, Jose R; Pongsiri, Montira J; Sanjur, Oris I; Pecor, James E; Auguste, Albert J; Kramer, Laura D

    2016-04-01

    Landscape changes occurring in Panama, a country whose geographic location and climate have historically supported arbovirus transmission, prompted the hypothesis that arbovirus prevalence increases with degradation of tropical forest habitats. Investigations at four variably degraded sites revealed a diverse array of potential mosquito vectors, several of which are known vectors of arbovirus pathogens. Overall, 675 pools consisting of 25,787 mosquitoes and representing 29 species from nine genera (collected at ground and canopy height across all habitats) were screened for cytopathic viruses on Vero cells. We detected four isolates of Gamboa virus (family:Bunyaviridae; genus:Orthobunyavirus) from pools of Aedeomyia squamipennis captured at canopy level in November 2012. Phylogenetic characterization of complete genome sequences shows the new isolates to be closely related to each other with strong evidence of reassortment among the M segment of Panamanian Gamboa isolates and several other viruses of this group. At the site yielding viruses, Soberanía National Park in central Panama, 18 mosquito species were identified, and the predominant taxa included A. squamipennis,Coquillettidia nigricans, and Mansonia titillans. PMID:26834200

  17. Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

    SciTech Connect

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury; Anthony, Karen; Ledizet, Michel; Modis, Yorgo

    2009-07-31

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  18. Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies

    SciTech Connect

    Nelson, Gretchen E.; Sisler, Jerry R.; Chandran, Dev; Moss, Bernard

    2008-10-25

    The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.

  19. Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin

    PubMed Central

    Tan, Gene S.; Lee, Peter S.; Hoffman, Ryan M. B.; Mazel-Sanchez, Beryl; Krammer, Florian; Leon, Paul E.; Ward, Andrew B.; Wilson, Ian A.

    2014-01-01

    ABSTRACT Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043. IMPORTANCE The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing

  20. Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling

    SciTech Connect

    Brunner, J.; Zugliani, C. ); Mischler, R. )

    1991-03-05

    Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions ({approximately}pH 5) this protein undergoes a conformational change that triggers the exposure of the fusion peptide, the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane ) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-(11-(4-(3-(trifluoromethyl)diazirinyl)phenyl)(2-{sup 3}H)undecanoyl)-sn-glycero-3-phosphocholine (({sup 3}H)PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed the authors to investigate both the interaction of viruses with the vesicles under perfusion conditions and the fusion process itself occurring at elevated temperatures only. Despite the observed binding of viruses to LUVs at pH 5 and 0C, labeling of HA2 was very weak. They have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles.

  1. Maturation-Dependent Human Immunodeficiency Virus Type 1 Particle Fusion Requires a Carboxyl-Terminal Region of the gp41 Cytoplasmic Tail▿

    PubMed Central

    Jiang, Jiyang; Aiken, Christopher

    2007-01-01

    Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), typically encode fusion glycoproteins with long cytoplasmic tails (CTs). We previously reported that immature HIV-1 particles are inhibited for fusion with target cells by a mechanism requiring the 152-amino-acid CT of gp41. The gp41 CT was also shown to mediate the detergent-resistant association of the HIV-1 envelope glycoprotein complex with immature HIV-1 particles, indicating that the gp41 CT forms a stable complex with Gag in immature virions. In the present study, we analyzed the effects of progressive truncations and point mutations in the gp41 CT on the fusion of mature and immature HIV-1 particles with target cells. We also determined the effects of these mutations on the detergent-resistant association of gp41 with immature HIV-1 particles. Removal of the C-terminal 28 amino acids relieved the dependence of HIV-1 fusion on maturation. However, a mutant Env protein lacking this region remained associated with immature HIV-1 particles treated with nonionic detergent. Further mutational analysis of the C-terminal region of gp41 revealed two specific sequences required for maturation-dependent HIV-1 fusion. Collectively, our results demonstrate that the extreme C terminus of gp41 plays a key role in coupling HIV-1 fusion competence to virion maturation. They further indicate that the stable association of gp41 with Gag in immature virions is not sufficient for inhibition of immature HIV-1 particle fusion. PMID:17609279

  2. The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain

    PubMed Central

    Ito, Morihiro; Ohtsuka, Junpei; Hara, Kenichiro; Komada, Hiroshi; Nishio, Machiko; Nosaka, Tetsuya

    2015-01-01

    ABSTRACT Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular

  3. Progressive Truncations C Terminal to the Membrane-Spanning Domain of Simian Immunodeficiency Virus Env Reduce Fusogenicity and Increase Concentration Dependence of Env for Fusion

    PubMed Central

    Lin, Xiaoxu; Derdeyn, Cynthia A.; Blumenthal, Robert; West, John; Hunter, Eric

    2003-01-01

    The simian immunodeficiency virus (SIV) transmembrane (TM) protein, gp41, has multiple functions, which include anchoring the glycoprotein complex in the lipid envelope of the virus and mediating fusion of the virus and host cell membranes. Recently, a series of mutants of the SIVmac239 TM protein that have truncations at the carboxyl terminus of the membrane-spanning domain (MSD) have been characterized (J. T. West, P. Johnston, S. R. Dubay, and E. Hunter, J. Virol. 75:9601-9612, 2001). These mutants retained membrane anchorage but demonstrated reduced fusogenicity and infectivity as the MSD length was shortened. We have established a novel three-color fluorescence assay, which allows qualitative confocal and quantitative flow cytometric analyses, to further characterize the nature of the fusion defect in five of the MSD mutants: TM185, TM186, TM187, TM188, and TM189. Our analysis showed that each mutant could mediate complete lipid and aqueous dye transfer at early time points after effector and target cell mixing. No hemifusion with only lipid dye flux was detected. However, another intermediate fusion stage, which appears to involve small-fusion-pore formation that allowed small aqueous dye transfer but prevented the exchange of large cytoplasmic components, was identified infrequently in mutant-Env-expressing cell and target cell mixtures. Quantitative flow cytometric analysis of these mutants demonstrated that the TM187, TM188, and TM189 mutants were significantly more fusogenic than TM185 and TM186 but remained significantly impaired compared to the wild type. Moreover, fusion efficiency showed an increased dependence on the expression level of glycoproteins, suggesting that, for these mutants, formation of an active fusion complex was an increasingly stochastic event. PMID:12768026

  4. Anti-viral activity of single-stranded homopolynucleotides against encephalomyocarditis virus and Semliki Forest virus in adult mice without interferon induction.

    PubMed

    Stebbing, N; Grantham, C A; Carey, N H

    1976-01-01

    Single administrations of poly C or poly I are anti-viral against infections of encephalomyocarditis (EMC) and Semliki Forest virus (SFV) in mice but poly U and poly A are not. The degree of protection is dose-dependent and mice which die do so at a later time when untreated controls even in a strain of mouse in which the time of death is not dependent on the dose of virus given. No circulating interferon is found after treating mice with poly C or poly I even at polynucleotide doses which give the same degree of protection as the interferon inducer, poly I:C. Several additional features distinguish the protection by poly C and poly I from interferon induction: the effect is low 24h before infection and maximal 6 h before infection, the effect is short-lived and mice do not show hypo-reactivation to repeated treatment. Limited treatment of mice with poly I:C, interferon or poly C before infection itself results in additional protection when poly C is also administered after infection, indicating that poly C has an effect after onset of virus replication. After infection poly C and poly I are both more effective by the intravenous route but before infection they are most effective when administered by the same route as the virus. Quantitative comparisons of the protective effects of poly C, poly I and the interferon inducer, poly I:C, are possible from dose response curves of the polynucleotides at different times relative to infection and by different routes of administration. The results are considered in relation to the presence of homopolyribonucleotide tracts in the viral genomes and effects on the reticulo-endothelial system of the mice. PMID:173799

  5. Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine

    PubMed Central

    Mackow, Natalie; Amaro-Carambot, Emérito; Liang, Bo; Surman, Sonja; Lingemann, Matthias; Yang, Lijuan; Collins, Peter L.

    2015-01-01

    ABSTRACT Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously described attenuating mutation that was stabilized against deattenuation, specifically, a non-temperature-sensitive deletion mutation involving 6 nucleotides in the overlapping P/C open reading frames (ORFs) (CΔ170) or a temperature-sensitive missense mutation in the L ORF (LY942A). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as the final titer of wild-type (wt) HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-CΔ170-F1, -F2, and -F3 and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-CΔ170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates, highly restricted in lungs, and genetically stable in vivo. Among the CΔ170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of overattenuation. The CΔ170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. IMPORTANCE There are no vaccines for the pediatric respiratory pathogens RSV and HPIV. We are developing live attenuated RSV and HPIV vaccines for use in virus-naive infants. Live attenuated RSV strains in particular are difficult to develop due to their poor growth and physical instability, but these obstacles could be

  6. Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

    PubMed Central

    Varjak, Margus; Saul, Sirle; Arike, Liisa; Lulla, Aleksei; Peil, Lauri

    2013-01-01

    Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection. PMID:23864636

  7. Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer▿

    PubMed Central

    Chaiwatpongsakorn, Supranee; Epand, Raquel F.; Collins, Peter L.; Epand, Richard M.; Peeples, Mark E.

    2011-01-01

    The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni+2 column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming “hat pin”-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide. PMID:21307202

  8. A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea Virus In Vitro

    PubMed Central

    Hashish, Emad A.; Zhang, Chengxian; Ruan, Xiaosai; Knudsen, David E.; Chase, Christopher C.; Isaacson, Richard E.; Zhou, Guoqiang

    2013-01-01

    Diarrhea is one of the most important bovine diseases. Enterotoxigenic Escherichia coli (ETEC) and bovine viral diarrhea virus (BVDV) are the major causes of diarrhea in calves and cattle. ETEC expressing K99 (F5) fimbriae and heat-stable type Ia (STa) toxin are the leading bacteria causing calf diarrhea, and BVDV causes diarrhea and other clinical illnesses in cattle of all ages. It is reported that maternal immunization with K99 fimbrial antigens provides passive protection to calves against K99 fimbrial ETEC and that BVDV major structural protein E2 elicits antibodies neutralizing against BVDV viral infection. Vaccines inducing anti-K99 and anti-STa immunity would protect calves more effectively against ETEC diarrhea, and those also inducing anti-E2 neutralizing antibodies would protect calves and cattle against diarrhea caused by both ETEC and BVDV. In this study, we used the ETEC K99 major subunit FanC as a backbone, genetically embedded the STa toxoid STaP12F and the most-antigenic B-cell epitope and T-cell epitope predicted from the BVDV E2 glycoprotein into FanC for the multivalent antigen FanC-STa-E2, and examined immunogenicity of this multivalent antigen to assess vaccine potential against bovine diarrhea. Mice intraperitoneally (i.p.) immunized with this multivalent antigen developed anti-K99, anti-STa, and anti-BVDV antibodies. Moreover, elicited antibodies showed neutralization activities, as they inhibited adherence of K99 fimbrial E. coli, neutralized STa toxin, and prevented homologous BVDV viral infection in vitro. Results from this study suggest that this multiepitope fusion antigen can potentially be developed as a vaccine for broad protection against bovine diarrhea and that the multiepitope fusion strategy may be generally applied for multivalent vaccine development against heterogeneous pathogens. PMID:23697572

  9. A multiepitope fusion antigen elicits neutralizing antibodies against enterotoxigenic Escherichia coli and homologous bovine viral diarrhea virus in vitro.

    PubMed

    Hashish, Emad A; Zhang, Chengxian; Ruan, Xiaosai; Knudsen, David E; Chase, Christopher C; Isaacson, Richard E; Zhou, Guoqiang; Zhang, Weiping

    2013-07-01

    Diarrhea is one of the most important bovine diseases. Enterotoxigenic Escherichia coli (ETEC) and bovine viral diarrhea virus (BVDV) are the major causes of diarrhea in calves and cattle. ETEC expressing K99 (F5) fimbriae and heat-stable type Ia (STa) toxin are the leading bacteria causing calf diarrhea, and BVDV causes diarrhea and other clinical illnesses in cattle of all ages. It is reported that maternal immunization with K99 fimbrial antigens provides passive protection to calves against K99 fimbrial ETEC and that BVDV major structural protein E2 elicits antibodies neutralizing against BVDV viral infection. Vaccines inducing anti-K99 and anti-STa immunity would protect calves more effectively against ETEC diarrhea, and those also inducing anti-E2 neutralizing antibodies would protect calves and cattle against diarrhea caused by both ETEC and BVDV. In this study, we used the ETEC K99 major subunit FanC as a backbone, genetically embedded the STa toxoid STaP12F and the most-antigenic B-cell epitope and T-cell epitope predicted from the BVDV E2 glycoprotein into FanC for the multivalent antigen FanC-STa-E2, and examined immunogenicity of this multivalent antigen to assess vaccine potential against bovine diarrhea. Mice intraperitoneally (i.p.) immunized with this multivalent antigen developed anti-K99, anti-STa, and anti-BVDV antibodies. Moreover, elicited antibodies showed neutralization activities, as they inhibited adherence of K99 fimbrial E. coli, neutralized STa toxin, and prevented homologous BVDV viral infection in vitro. Results from this study suggest that this multiepitope fusion antigen can potentially be developed as a vaccine for broad protection against bovine diarrhea and that the multiepitope fusion strategy may be generally applied for multivalent vaccine development against heterogeneous pathogens. PMID:23697572

  10. EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression

    PubMed Central

    Stobart, Christopher C.; Hotard, Anne L.; Villenave, Remi; Meng, Jia; Pretto, Carla D.; Shields, Michael D.; Nguyen, Minh Trang; Todd, Sean O.; Chi, Michael H.; Hammonds, Jason; Krumm, Stefanie A.; Spearman, Paul; Plemper, Richard K.; Sakamoto, Kaori; Peebles, R. Stokes; Power, Ultan F.; Moore, Martin L.

    2016-01-01

    Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2–20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2–20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2–20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2–20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2–20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease. PMID:27152417