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Jose A Melero - One of the best experts on this subject based on the ideXlab platform.
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correction corrigendum potent single domain antibodies that arrest respiratory syncytial Virus Fusion Protein in its preFusion state
Nature Communications, 2017Co-Authors: Iebe Rossey, Vicente Mas, Morgan S A Gilman, Stephanie C Kabeche, Koen Sedeyn, Daniel Wrapp, Masaru Kanekiyo, Man Chen, Jan Spitaels, Jose A MeleroAbstract:Nature Communications 8: Article number: 14158 (2017); Published 13 February 2017; Updated 29 November 2017. In this Article, the clinical isolate MAD/GM3_10/14 of respiratory syncytial Virus (RSV) is incorrectly described as an RSV A isolate. MAD/GM3_10/14 has been identified as an RSV B isolate onthe basis of its Fusion Protein sequence.
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recombinant sendai Viruses expressing Fusion Proteins with two furin cleavage sites mimic the syncytial and receptor independent infection properties of respiratory syncytial Virus
Journal of Virology, 2011Co-Authors: Joanna Rawling, Olga Cano, Dominique Garcin, Daniel Kolakofsky, Jose A MeleroAbstract:Cell entry by paramyxoViruses requires Fusion between viral and cellular membranes. ParamyxoVirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of Fusion are mediated by the viral Fusion (F) Protein, which requires proteolytic processing at a basic cleavage site in order to be active for Fusion. In common with most paramyxoViruses, Fusion mediated by Sendai Virus F Protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) Protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial Virus Fusion Protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) Protein. Moreover, F(RSV) is unique among paramyxoVirus Fusion Proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment Protein for syncytium formation in transfected cells. We now describe recombinant Sendai Viruses (rSeV) that express mutant F Proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant Viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment Protein.
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insertion of the two cleavage sites of the respiratory syncytial Virus Fusion Protein in sendai Virus Fusion Protein leads to enhanced cell cell Fusion and a decreased dependency on the hn attachment Protein for activity
Journal of Virology, 2008Co-Authors: Joanna Rawling, Blanca Garciabarreno, Jose A MeleroAbstract:Cell entry by paramyxoViruses requires Fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral Fusion (F) glycoProtein and usually requires the aid of the attachment glycoProtein (G, H or HN, depending on the Virus). Human respiratory syncytial Virus F Protein (FRSV) is able to mediate membrane Fusion in the absence of the attachment G Protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell Fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai Virus F Protein (FSeV), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment Protein and trypsin in order to fuse cells. Inclusion of both FRSV cleavage sites in FSeV resulted in a dramatic increase in cell-cell Fusion activity in the presence of HN. Furthermore, chimeric FSeV mutants containing both FRSV cleavage sites demonstrated cell-cell Fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxoVirus F Protein for cell-cell Fusion in the absence of an attachment Protein.
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thermostability of the human respiratory syncytial Virus Fusion Protein before and after activation implications for the membrane Fusion mechanism
Journal of General Virology, 2004Co-Authors: Begona M Ruizarguello, Olga Cano, Blanca Garciabarreno, L J Calder, Steve A Wharton, John J Skehel, Diana Martin, S R Martin, Miguel Calero, Jose A MeleroAbstract:Anchorless Fusion (F) Proteins () of human respiratory syncytial Virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-Fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the Fusion peptide, a deletion mutant of lacking the first half of the Fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the Fusion peptide. As exposure of the Fusion peptide is a key event that occurs after activation of F Proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F Protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral Proteins involved in membrane Fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane Fusion mediated by RSV F Protein.
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cleavage of the human respiratory syncytial Virus Fusion Protein at two distinct sites is required for activation of membrane Fusion
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Luis Gonzalezreyes, Blanca Garciabarreno, L J Calder, John J Skehel, Don C Wiley, Begona M Ruizarguello, Juan Antonio Lopez, Juan Pablo Albar, Jose A MeleroAbstract:Preparations of purified full-length Fusion (F) Protein of human respiratory syncytial Virus (HRSV) expressed in recombinant vaccinia-F infected cells, or of an anchorless mutant (FTM−) lacking the C-terminal 50 amino acids secreted from vaccinia-FTM−-infected cells contain a minor polypeptide that is an intermediate product of proteolytic processing of the F Protein precursor F0. N-terminal sequencing of the intermediate demonstrated that it is generated by cleavage at a furin-motif, residues 106–109 of the F sequence. By contrast, the F1 N terminus derives from cleavage at residue 137 of F0 which is also C-terminal to a furin recognition site at residues 131–136. Site-directed mutagenesis indicates that processing of F0 Protein involves independent cleavage at both sites. Both cleavages are required for the F Protein to be active in membrane Fusion as judged by syncytia formation, and they allow changes in F structure from cone- to lollipop-shaped spikes and the formation of rosettes by anchorless F.
Rebecca Ellis Dutch - One of the best experts on this subject based on the ideXlab platform.
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mutations in the transmembrane domain and cytoplasmic tail of hendra Virus Fusion Protein disrupt Virus like particle assembly
Journal of Virology, 2017Co-Authors: Nicolas Cifuentesmunoz, Rebecca Ellis Dutch, Stacy R Webb, Weina Sun, Greeshma Ray, Phuong Tieu Schmitt, Kathleen Marie Gibson, Anthony P SchmittAbstract:Hendra Virus (HeV) is a zoonotic paramyxoVirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral Fusion Protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F Proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of Virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F Protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F Protein was competent for endocytic trafficking. The F and M Proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these Proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra Virus and Nipah Virus are zoonotic paramyxoViruses that cause lethal infections in humans. Unlike that for most paramyxoViruses, activation of the henipaVirus Fusion Protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra Virus F Protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipaVirus assembly process and provide a novel model for the interplay between glycoProtein trafficking and paramyxoVirus assembly.
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hendra Virus Fusion Protein transmembrane domain contributes to pre Fusion Protein stability
Journal of Biological Chemistry, 2017Co-Authors: Stacy R Webb, Michael Fried, Tamas Nagy, Hunter N B Moseley, Rebecca Ellis DutchAbstract:Enveloped Viruses utilize Fusion (F) Proteins studding the surface of the Virus to facilitate membrane Fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the Virus can ultimately reproduce and spread. To drive Fusion, the F Protein undergoes an irreversible conformational change, transitioning from a metastable pre-Fusion conformation to a more thermodynamically stable post-Fusion structure. Understanding the elements that control stability of the pre-Fusion state and triggering to the post-Fusion conformation is important for understanding F Protein function. Mutations in F Protein transmembrane (TM) domains implicated the TM domain in the Fusion process, but the structural and molecular details in Fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra Virus F Protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726–35735). To determine factors driving this association, 140 paramyxoVirus F Protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra Virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole Protein resulted in decreased Protein stability, including pre-Fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F Protein function and pre-Fusion stability.
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residues in the hendra Virus Fusion Protein transmembrane domain are critical for endocytic recycling
Journal of Virology, 2012Co-Authors: Andreea Popa, James R Carter, Stacy E Smith, Lance M Hellman, Michael Fried, Rebecca Ellis DutchAbstract: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.
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beyond anchoring the expanding role of the hendra Virus Fusion Protein transmembrane domain in Protein folding stability and function
Journal of Virology, 2012Co-Authors: Everett Clinton Smith, Lance M Hellman, Michael Fried, Megan R Culler, Trevor P Creamer, Rebecca Ellis DutchAbstract: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.
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a mature and fusogenic form of the nipah Virus Fusion Protein requires proteolytic processing by cathepsin l
Virology, 2006Co-Authors: Cara T Pager, Willie Warren Craft, Jared R Patch, Rebecca Ellis DutchAbstract:The Nipah Virus Fusion (F) Protein is proteolytically processed to F1 + F2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and Fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah Virus F-mediated Fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored Fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F Protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.
Trudy G. Morrison - One of the best experts on this subject based on the ideXlab platform.
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thiol disulfide exchange is required for membrane Fusion directed by the newcastle disease Virus Fusion Protein
Journal of Virology, 2007Co-Authors: Surbhi Jain, Lori W. Mcginnes, Trudy G. MorrisonAbstract:Newcastle disease Virus (NDV), an avian paramyxoVirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) Protein to sialic acid-containing receptors, followed by Fusion of viral and cell membranes, which is mediated by the Fusion (F) Protein. Like all class 1 viral Fusion Proteins, the paramyxoVirus F Protein is thought to undergo dramatic conformational changes upon activation. How the F Protein accomplishes extensive conformational rearrangements is unclear. Since several viral Fusion Proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV Proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity—5′5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-Protein disulfide isomerase antibody—inhibited cell-cell Fusion and Virus entry but had no effect on cell viability, glycoProtein surface expression, or HN Protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F Protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F Protein, but not HN Protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell Fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F Protein are reduced by the Protein disulfide isomerase family of isomerases and that F Protein exists as a mixture of oxidized and reduced forms. In the presence of HN Protein, only the reduced form may proceed to refold into additional intermediates, leading to the Fusion of membranes.
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mutational analysis of the membrane proximal heptad repeat of the newcastle disease Virus Fusion Protein
Virology, 2001Co-Authors: Lori W. Mcginnes, Theresa Sergel, Hong Chen, Ludwig Hamo, Steve Schwertz, Trudy G. MorrisonAbstract:ParamyxoVirus Fusion Proteins have two heptad repeat domains, HR1 and HR2, that have been implicated in the Fusion activity of the Protein. Peptides from these two domains form a six-stranded, coiled-coil with the HR1 sequences forming a central trimer and three molecules of the HR2 helix located within the grooves in the central trimer (Baker et al., 1999, Mol. Cell 3, 309; Zhao et al. 2000, Proc. Natl. Acad. Sci. USA 97, 14172). Nonconservative mutations were made in the HR2 domain of the Newcastle disease Virus Fusion Protein in residues that are likely to form contacts with the HR1 core trimer. These residues form the hydrophobic face of the helix and adjacent residues ("a" and "g" positions in the HR2 helical wheel structure). Mutant Proteins were characterized for effects on synthesis, steady-state levels, proteolytic cleavage, and surface expression as well as Fusion activity as measured by syncytia formation, content mixing, and lipid mixing. While all mutant Proteins were transport competent and proteolytically cleaved, these mutations did variously affect Fusion activity of the Protein. Nonconservative mutations in the "g" position had no effect on Fusion. In contrast, single changes in the middle "a" position of HR2 inhibited lipid mixing, content mixing, and syncytia formation. A single mutation in the more carboxyl-terminal "a" position had minimal effects on lipid mixing but did inhibit content mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved in posttranslational interactions with HR1 that mediate the close approach of membranes. These results also suggest that the HR2 domain, particularly the carboxyl-terminal region, plays an additional role in Fusion, a role related to content mixing and syncytia formation.
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mutations in the Fusion peptide and adjacent heptad repeat inhibit folding or activity of the newcastle disease Virus Fusion Protein
Journal of Virology, 2001Co-Authors: Theresa Sergel, Lori W. Mcginnes, Trudy G. MorrisonAbstract:ParamyxoVirus Fusion Proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the Fusion activity of the Protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172–14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease Virus Fusion Protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the “a” and “d” positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant Proteins expressed on the cell surface were characterized for Fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the “a” position interfered with proteolytic cleavage and surface expression of the Protein, implicating the HR1 domain in the folding of the F Protein. However, mutation of five of seven “d” position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the Fusion activity of the Protein. One of these “d” mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most “d” position residues do affect Fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal Fusion activity. That most of the “d” position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the preFusion form of the Protein. We also found that mutations within the Fusion peptide, at residue 128, can interfere with folding of the Protein, implicating this region in folding of the molecule. No characterized mutation enhanced Fusion.
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interaction of peptides with sequences from the newcastle disease Virus Fusion Protein heptad repeat regions
Journal of Virology, 1999Co-Authors: John K Young, Matthew C Abramowitz, Trudy G. MorrisonAbstract:Typical of many viral Fusion Proteins, the sequence of the Newcastle disease Virus (NDV) Fusion Protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the Fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited Fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the Fusion Protein. This inhibition was Virus specific since the peptide had minimal effect on Fusion directed by the Sendai Virus glycoProteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized Fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit Fusion, equimolar mixtures of the two peptides had minimal effect on Fusion, suggesting that the two peptides form a complex preventing their interaction with a target Protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix.
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mutations in the Fusion peptide and heptad repeat regions of the newcastle disease Virus Fusion Protein block Fusion
Journal of Virology, 1994Co-Authors: T Sergelgermano, Cathy Mcquain, Trudy G. MorrisonAbstract:Nonconservative mutations were introduced by site-specific mutagenesis into the Fusion peptide and the adjacent heptad repeat region of the Fusion Protein of Newcastle disease Virus in order to determine the role of both regions in the Fusion activity of the Protein. Mutations in both regions that allowed for proper folding and intracellular transport of the Protein blocked the Fusion activity of the Protein when assayed in the presence of the hemagglutinin-neuraminidase Protein.
Margaret Kielian - One of the best experts on this subject based on the ideXlab platform.
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E1 mutants identify a critical region in the trimer interface of the Semliki Forest Virus Fusion Protein
2016Co-Authors: Catherine Y. Liu, Margaret KielianAbstract:The alphaVirus Semliki Forest Virus (SFV) uses a membrane Fusion reaction to infect host cells. Fusion of the Virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane Protein E1. During Fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 homotrimer is critical to membrane Fusion, but the mechanism of tri-merization is not understood. The crystal structure of the postFusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphaVirus E1 sequences. We tested the contribution of this amino acid to trimerization and Fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV Fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and Fusion required a lower pH. While there are extensive contacts between E1 subunits in the homotrimer, the D188K mutant identifies an important “hot spot ” for Protein-Protein interactions within the core trimer. In an aqueous environment, phospholipid bilayers are stabl
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role of conserved histidine residues in the low ph dependence of the semliki forest Virus Fusion Protein
Journal of Virology, 2009Co-Authors: Yan Zheng, Zhao Ling Qin, Margaret KielianAbstract:A wide variety of enveloped Viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger Virus-membrane Fusion. For alphaViruses such as Semliki Forest Virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric Virus envelope Proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane Fusion Protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the Fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create Virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of Virus-membrane Fusion. By contrast, the E1 H3A mutation produced impaired Virus growth and a markedly more acidic pH requirement for Virus-membrane Fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 Protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane Fusion.
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novel mutations that control the sphingolipid and cholesterol dependence of the semliki forest Virus Fusion Protein
Journal of Virology, 2002Co-Authors: Prodyot K Chatterjee, Christina H Eng, Margaret KielianAbstract:The enveloped alphaVirus Semliki Forest Virus (SFV) infects cells via a membrane Fusion reaction mediated by the E1 membrane Protein. Efficient SFV-membrane Fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 Protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane Fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient Fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the Fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in Virus-membrane Fusion.
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membrane and Protein interactions of a soluble form of the semliki forest Virus Fusion Protein
Journal of Virology, 1994Co-Authors: Matthew R Klimjack, Susan Jeffrey, Margaret KielianAbstract:Semliki Forest Virus is an enveloped alphaVirus that infects cells by a membrane Fusion reaction triggered by the low pH present in endocytic vacuoles. Fusion is mediated by the E1 spike Protein subunit. During Fusion, several conformational changes occur in E1 and E2, the two transmembrane subunits of the spike Protein. These changes include dissociation of the E1-E2 dimer, alteration of the trypsin sensitivity and monoclonal antibody binding patterns of E1, and formation of a sodium dodecyl sulfate (SDS)-resistant E1 homotrimer. A critical characteristic of Semliki Forest Virus Fusion is also its dependence on the presence of both cholesterol and sphingomyelin in the target membrane. We have here examined the conformational changes induced by low pH treatment of E1*, the water-soluble, proteolytically truncated ectodomain of the E1 subunit. Following low pH treatment, E1* was shown to bind efficiently to artificial liposomes. Similar to Virus Fusion, optimal E1*-liposome binding required low pH, cholesterol, and sphingomyelin. The E1 ectodomain, although monomeric in its neutral pH form, assembled into an SDS-resistant oligomer following treatment at low pH. This low pH-induced oligomerization required target membranes containing both cholesterol and sphingomyelin. Our results demonstrate that the E1 ectodomain responds to low pH similarly to the full-length E1 subunit. The ectodomain facilitates the characterization of conformational changes and membrane binding in the absence of Virus Fusion or other Virus components.
Mark E Peeples - One of the best experts on this subject based on the ideXlab platform.
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five residues in the apical loop of the respiratory syncytial Virus Fusion Protein f2 subunit are critical for its Fusion activity
Journal of Virology, 2018Co-Authors: Stephanie N Hicks, Supranee Chaiwatpongsakorn, Heather M Costello, Jason S Mclellan, Mark E Peeples, Stephanie N HicksAbstract:The respiratory syncytial Virus (RSV) Fusion (F) Protein is a trimeric, membrane-anchored glycoProtein capable of mediating both Virus-target cell membrane Fusion to initiate infection and cell-cell Fusion, even in the absence of the attachment glycoProtein. The F Protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy preFusion conformation. To mediate Fusion, the F Protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the Protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for Fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered Fusion following a forced Fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent Fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F Protein. IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the Fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F Protein and, as such, may be ideal targets for antiviral drug development.
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soluble respiratory syncytial Virus Fusion Protein in the fully cleaved pretriggered state is triggered by exposure to low molarity buffer
Journal of Virology, 2011Co-Authors: Peter L Collins, Supranee Chaiwatpongsakorn, Mark E Peeples, Raquel F EpandAbstract: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.
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intracellular maturation of the newcastle disease Virus Fusion Protein is affected by strain differences in the predicted amphipathic α helix adjacent to the Fusion domain
Virology, 1995Co-Authors: Can Wang, Mark E PeeplesAbstract:Abstract The Fusion (F) glycoProtein of Newcastle disease Virus (NDV) contains a predicted amphipathic α-helix C-terminal to its Fusion domain. Of the 13 available NDV F Protein sequences, only the Australia-Victoria (AV) strain α-helix is weakened, by the replacement of Ala 159 with Thr. In this report, we demonstrate that the efficiency of cleavage and virion incorporation of the AV F Protein, unlike that of other strains, is temperature sensitive. Pulse/chase experiments at 42° revealed disulfidelinked aggregates containing both the F and hemagglutinin-neuraminidase glycoProteins in strain AV, but not in strain Beaudette C. Furthermore, a revertant derived from AV, whose helix-weakening Thr159 has been replaced with the consensus Ala, produced fewer F Protein aggregates, confirming the structural importance of this region in maturation. In addition, a novel disulfide-defined folding intermediate of the F Protein was detected.
