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Robert A. Lamb - One of the best experts on this subject based on the ideXlab platform.
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fusion activation by a headless parainfluenza virus 5 hemagglutinin neuraminidase stalk suggests a modular mechanism for triggering
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Sayantan Bose, Brett D Welch, Theodore S Jardetzky, Aarohi Zokarkar, George P Leser, Robert A. LambAbstract:The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoProteins. The receptor-binding Protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion Protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus–cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN Protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN Protein, we propose a simple model for the activation of paramyxovirus fusion.
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domain architecture and oligomerization properties of the paramyxovirus piv 5 hemagglutinin neuraminidase HN Protein
Virology, 2008Co-Authors: Ping Yuan, Robert A. Lamb, George P Leser, Borries Demeler, Theodore S JardetzkyAbstract:The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) Protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high α-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.
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The Signal for Clathrin-Mediated Endocytosis of the Paramyxovirus SV5 HN Protein Resides at the Transmembrane Domain–Ectodomain Boundary Region
Virology, 1999Co-Authors: George P Leser, Karen J. Ector, Margaret A. Shaughnessy, Robert A. LambAbstract:The hemagglutinin-neuraminidase (HN) glycoProtein of the paramyxovirus SV5 is internalized from the cell surface via clathrin-coated pits. However, the cytoplasmic domain of SV5 HN does not contain a previously characterized internalization motif. A cell-surface-expressed chimeric Protein (APK), consisting of the cytoplasmic tail, transmembrane (TM) domain, and 12 residues of the ectodomain of HN joined to the cytoplasmic Protein pyruvate kinase is internalized, indicating that the N-terminal region of HN contains an internalization signal. Although SV5 HN is internalized at a rate similar to that of influenza virus hemagglutinin (HA) mutant Y543, which contains a degenerate tyrosine-based signal in its cytoplasmic tail, the elimination of the majority of the HN cytoplasmic tail, or substitution of the HN TM domain with leucine residues, did not affect the rate of HN internalization. The HN Protein of the closely related virus, Newcastle disease virus (NDV), is not internalized from the cell surface. Working under the usual convention that the TM domain consists of the hydrophobic residues bounded by two charged residues, analysis of internalization of mutant and chimeric NDV HN molecules indicates that the first seven SV5 HN ectodomain residues are critical for internalization of HN. A glutamic acid residue (E37) that abuts this presumptive HN TM domain/ectodomain boundary is important for SV5 HN internalization.
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the signal for clathrin mediated endocytosis of the paramyxovirus sv5 HN Protein resides at the transmembrane domain ectodomain boundary region
Virology, 1999Co-Authors: George P Leser, Robert A. Lamb, Karen J. Ector, Margaret A. ShaughnessyAbstract:The hemagglutinin-neuraminidase (HN) glycoProtein of the paramyxovirus SV5 is internalized from the cell surface via clathrin-coated pits. However, the cytoplasmic domain of SV5 HN does not contain a previously characterized internalization motif. A cell-surface-expressed chimeric Protein (APK), consisting of the cytoplasmic tail, transmembrane (TM) domain, and 12 residues of the ectodomain of HN joined to the cytoplasmic Protein pyruvate kinase is internalized, indicating that the N-terminal region of HN contains an internalization signal. Although SV5 HN is internalized at a rate similar to that of influenza virus hemagglutinin (HA) mutant Y543, which contains a degenerate tyrosine-based signal in its cytoplasmic tail, the elimination of the majority of the HN cytoplasmic tail, or substitution of the HN TM domain with leucine residues, did not affect the rate of HN internalization. The HN Protein of the closely related virus, Newcastle disease virus (NDV), is not internalized from the cell surface. Working under the usual convention that the TM domain consists of the hydrophobic residues bounded by two charged residues, analysis of internalization of mutant and chimeric NDV HN molecules indicates that the first seven SV5 HN ectodomain residues are critical for internalization of HN. A glutamic acid residue (E37) that abuts this presumptive HN TM domain/ectodomain boundary is important for SV5 HN internalization.
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membrane fusion promoted by increasing surface densities of the paramyxovirus f and HN Proteins comparison of fusion reactions mediated by simian virus 5 f human parainfluenza virus type 3 f and influenza virus ha
Journal of Virology, 1998Co-Authors: Rebecca Ellis Dutch, Sangeeta Bagai Joshi, Robert A. LambAbstract:The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) Proteins and hemagglutinin-neuraminidase (HN) Proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F Protein but independent of the density of SV5 HN Protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F Protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN Protein, suggesting that the HPIV-3 HN Protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F Protein surface densities, suggesting that multiple fusion pores can be active during SV5 F Protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin Protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion Proteins.
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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inhibition of receptor binding stabilizes newcastle disease virus HN and f Protein containing complexes
Journal of Virology, 2006Co-Authors: Lori W. Mcginnes, Trudy G. MorrisonAbstract:Receptor binding of paramyxovirus attachment Proteins and the interactions between attachment and fusion (F) Proteins are thought to be central to activation of the F Protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F Protein interactions and HN Protein attachment to sialic acid receptors, HN and F Protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN Protein receptor binding, cells transfected with HN and F Protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F Protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F Protein-expressing cells resulted in a twofold decrease in amounts of HN and F Protein complexes. In contrast, high levels of complexes containing HN Protein and an uncleaved F Protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F Proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F Protein cleavage are necessary for disassociation of the HN and F Protein-containing complexes.
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Interacting domains of the HN and F Proteins of newcastle disease virus.
Journal of virology, 2003Co-Authors: Kathryn A. Gravel, Trudy G. MorrisonAbstract:The activation of most paramyxovirus fusion Proteins (F Proteins) requires not only cleavage of F0 to F1 and F2 but also coexpression of the homologous attachment Protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H Protein coexpression strongly suggests that an interaction between HN and F Proteins is required for fusion, and studies of chimeric HN Proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F Protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN Protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion Proteins containing these HN Protein sequences but not to GST or to GST containing HN Protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN Protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant Proteins. These mutations inhibited the fusion promotion activities of the Proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion Protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN Protein binds to the F Protein HR2 domain, an interaction important for the fusion promotion activity of the HN Protein.
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Newcastle Disease Virus HN Protein Alters the Conformation of the F Protein at Cell Surfaces
Journal of virology, 2002Co-Authors: Lori W. Mcginnes, Kathryn A. Gravel, Trudy G. MorrisonAbstract:Conformational changes in the Newcastle disease virus (NDV) fusion (F) Protein during activation of fusion and the role of HN Protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F Protein HR1 domain. This antibody immunoprecipitated both F0 and F1 forms of the fusion Protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN Protein. In marked contrast, this antibody detected significant conformational differences in the F Protein at cell surfaces, differences that depended upon HN Protein expression. The antibody minimally detected the F Protein, either cleaved or uncleaved, in the absence of HN Protein expression. However, when coexpressed with HN Protein, an uncleaved mutant F Protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN Protein. When the cleaved wild-type F Protein was coexpressed with HN Protein, the F Protein bound anti-HR1 antibody poorly although significantly more than F Protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F Proteins. This inhibition showed that fusion-competent F Proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F Protein is affected by HN Protein expression. Furthermore, the cleaved F Protein, when coexpressed with HN Protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN Protein attachment to receptors on red blood cells.
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A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion
Journal of virology, 2000Co-Authors: Theresa A. Sergel, Lori W. Mcginnes, Trudy G. MorrisonAbstract:The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F Protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the Protein or the fusion activity of the Protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these Proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant Protein was cleaved and expressed at the cell surface although the Protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A Protein was fusion inactive in the presence or absence of HN Protein expression. Mutant L289A Protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this Protein mediated syncytium formation in the absence of HN Protein expression although HN Protein enhanced fusion activity. These results show that a single amino acid change in the F1 portion of the NDV F Protein can alter the stringent requirement for HN Protein expression in syncytium formation.
Masato Tsurudome - One of the best experts on this subject based on the ideXlab platform.
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The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain
Journal of virology, 2015Co-Authors: Masato Tsurudome, Morihiro Ito, Machiko Nishio, Hiroshi Komada, Junpei Ohtsuka, Kenichiro Hara, Tetsuya NosakaAbstract:UNLABELLED 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 region of the head domain drastically change the F Protein specificity of the HN Protein, suggesting that the ability of a given HN Protein to interact with an F Protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN Proteins in triggering noncognate F Proteins.
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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
Journal of Virology, 2013Co-Authors: Masato Tsurudome, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Mito Nakahashi, Yoshiaki Matsushima, Tetsuya NosakaAbstract:ABSTRACT 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.
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Identification of Domains on the Fusion (F) Protein Trimer That Influence the Hemagglutinin-Neuraminidase Specificity of the F Protein in Mediating Cell-Cell Fusion
Journal of virology, 2011Co-Authors: Masato Tsurudome, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Mito Nakahashi, Tetsuya Nosaka, Yasuhiko ItoAbstract:For most paramyxoviruses, virus type-specific interaction between fusion (F) Protein and attachment Protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoProtein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F Proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F Protein contains the site(s) that determines its specificity for the HPIV2 HN Protein. In the present study, we further investigated the sites of the F Protein that could be critical for determining the HN Protein specificity. By analyzing the reported structure of the F Protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F Protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN Protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN Protein, suggesting that these domains are involved in determining the HN Protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN Protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.
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N-glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase Proteins of human parainfluenza virus type 4A and 4B.
Medical microbiology and immunology, 2000Co-Authors: Hiroshi Komada, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Masato Tsurudome, Shigeru Kusagawa, Hisataka Ohta, Myles O'brien, Hisanori Bando, Yasuhiko ItoAbstract:cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) Protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN Proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN Proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN Proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant Proteins, and found that the antigenicities of the mutant hPIV-4B HN Proteins were more similar to the hPIV-4A HN Protein than to the non-mutant hPIV-4B HN Protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes.
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Identification of regions on the fusion Protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase Proteins to promote cell fusion.
Journal of General Virology, 1998Co-Authors: Masato Tsurudome, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Shigeru Kusagawa, Kousuke Okamoto, Yasuhiko ItoAbstract:Using a plasmid expression system in HeLa cells, we have previously shown that the fusion (F) Protein of simian virus 41 (SV-41) induces cell fusion when coexpressed with the haemagglutinin-neuraminidase (HN) Protein of human parainfluenza virus type 2 (PIV-2), while the PIV-2 F Protein does not induce cell fusion with the SV-41 HN Protein. In the present study, we found that the PIV-2 F Protein induced extensive cell fusion with the HN Protein of mumps virus (MuV), whereas the SV-41 F Protein did not. Chimaeric analyses of the F Proteins of PIV-2 and SV-41 identified two regions (designated M1 and M2) on the PIV-2 F Protein, either of which was necessary for chimaeric F Proteins to show fusogenic activity with the MuV HN Protein. Subsequently, two additional regions (P1 and P2) were identified on the PIV-2 F Protein, both of which were necessary for chimaeric F Proteins to prevent induction of cell fusion with the SV-41 HN Protein. Consequently, it was proved that a given chimaeric F Protein, harbouring regions P1 and P2 together with either of region M1 or M2, induced cell fusion specifically with HN Proteins of PIV-2 and MuV, the same as the PIV-2 F Protein. Region M2 was located at the membrane proximal end of the PIV-2 F1 ectodomain, while regions P1, M1 and P2 clustered together in the middle of the ectodomain. These regions on the PIV-2 F Protein may be involved in a putative functional interaction with HN Proteins, which is considered to be a prerequisite for cell fusion.
Lori W. Mcginnes - 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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inhibition of receptor binding stabilizes newcastle disease virus HN and f Protein containing complexes
Journal of Virology, 2006Co-Authors: Lori W. Mcginnes, Trudy G. MorrisonAbstract:Receptor binding of paramyxovirus attachment Proteins and the interactions between attachment and fusion (F) Proteins are thought to be central to activation of the F Protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F Protein interactions and HN Protein attachment to sialic acid receptors, HN and F Protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN Protein receptor binding, cells transfected with HN and F Protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F Protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F Protein-expressing cells resulted in a twofold decrease in amounts of HN and F Protein complexes. In contrast, high levels of complexes containing HN Protein and an uncleaved F Protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F Proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F Protein cleavage are necessary for disassociation of the HN and F Protein-containing complexes.
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Newcastle Disease Virus HN Protein Alters the Conformation of the F Protein at Cell Surfaces
Journal of virology, 2002Co-Authors: Lori W. Mcginnes, Kathryn A. Gravel, Trudy G. MorrisonAbstract:Conformational changes in the Newcastle disease virus (NDV) fusion (F) Protein during activation of fusion and the role of HN Protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F Protein HR1 domain. This antibody immunoprecipitated both F0 and F1 forms of the fusion Protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN Protein. In marked contrast, this antibody detected significant conformational differences in the F Protein at cell surfaces, differences that depended upon HN Protein expression. The antibody minimally detected the F Protein, either cleaved or uncleaved, in the absence of HN Protein expression. However, when coexpressed with HN Protein, an uncleaved mutant F Protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN Protein. When the cleaved wild-type F Protein was coexpressed with HN Protein, the F Protein bound anti-HR1 antibody poorly although significantly more than F Protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F Proteins. This inhibition showed that fusion-competent F Proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F Protein is affected by HN Protein expression. Furthermore, the cleaved F Protein, when coexpressed with HN Protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN Protein attachment to receptors on red blood cells.
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A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion
Journal of virology, 2000Co-Authors: Theresa A. Sergel, Lori W. Mcginnes, Trudy G. MorrisonAbstract:The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F Protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the Protein or the fusion activity of the Protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these Proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant Protein was cleaved and expressed at the cell surface although the Protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A Protein was fusion inactive in the presence or absence of HN Protein expression. Mutant L289A Protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this Protein mediated syncytium formation in the absence of HN Protein expression although HN Protein enhanced fusion activity. These results show that a single amino acid change in the F1 portion of the NDV F Protein can alter the stringent requirement for HN Protein expression in syncytium formation.
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Role of carbohydrate processing and calnexin binding in the folding and activity of the HN Protein of Newcastle disease virus
Virus research, 1998Co-Authors: Lori W. Mcginnes, Trudy G. MorrisonAbstract:The role of carbohydrate processing and calnexin binding in the folding pathway and activity of the hemagglutinin-neuraminidase (HN) Protein of Newcastle disease virus (NDV) was explored in infected cells using the inhibitor castanospermine (CST). Calnexin-HN Protein complexes were demonstrated by coimmunoprecipitation using antibody specific for calnexin or HN Protein. As in other systems, this complex was not detected in CST treated cells. In cells incubated in CST, the synthesis and stability of the HN Protein was unaffected. However, as monitored by the appearance of conformationally sensitive antigenic sites, the folding of the HN Protein in CST treated cells was approximately twice as slow than in untreated cells. This folding was ultimately efficient since there was no evidence for significant amounts of irreversibly aggregated forms which never acquired a mature conformation. Most significantly, the folding sequence as measured by the order of appearance of conformationally sensitive antigenic sites (McGinnes and Morrison, Virology 199, 255) was unaffected by CST. Thus while calnexin functions to speed the folding of the HN Protein, it is not required for the folding of this Protein. In addition, the Protein synthesized in the presence of CST had significant levels of neuraminidase and hemagglutination activity suggesting that processing of the carbohydrate has a minimal role in the activity of the Protein.
Yasuhiko Ito - One of the best experts on this subject based on the ideXlab platform.
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Identification of Domains on the Fusion (F) Protein Trimer That Influence the Hemagglutinin-Neuraminidase Specificity of the F Protein in Mediating Cell-Cell Fusion
Journal of virology, 2011Co-Authors: Masato Tsurudome, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Mito Nakahashi, Tetsuya Nosaka, Yasuhiko ItoAbstract:For most paramyxoviruses, virus type-specific interaction between fusion (F) Protein and attachment Protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoProtein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F Proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F Protein contains the site(s) that determines its specificity for the HPIV2 HN Protein. In the present study, we further investigated the sites of the F Protein that could be critical for determining the HN Protein specificity. By analyzing the reported structure of the F Protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F Protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN Protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN Protein, suggesting that these domains are involved in determining the HN Protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN Protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.
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N-glycosylation contributes to the limited cross-reactivity between hemagglutinin neuraminidase Proteins of human parainfluenza virus type 4A and 4B.
Medical microbiology and immunology, 2000Co-Authors: Hiroshi Komada, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Masato Tsurudome, Shigeru Kusagawa, Hisataka Ohta, Myles O'brien, Hisanori Bando, Yasuhiko ItoAbstract:cDNAs encoding human parainfluenza virus type 4B (hPIV-4B) hemagglutinin neuraminidase (HN) Protein were cloned and the nucleotide sequences were determined. A high degree of identity (81.4%) was observed between the nucleotide sequences of hPIV-4A and -4B HN Proteins, and an 87.3% identity was found between the deduced amino acid sequences. This degree of identity is considered to be greater than immunological similarity between hPIV-4A and -4B HN Proteins determined using monoclonal antibodies. To elucidate the causes of the antigenic difference between HN Proteins of hPIV-4A and -4B, we constructed three cDNAs of hPIV-4B HN whose potential N-glycosylation sites were partially or completely the same as in hPIV-4A HN cDNA. We compared the antigenicity of the expressed wild-type and mutant Proteins, and found that the antigenicities of the mutant hPIV-4B HN Proteins were more similar to the hPIV-4A HN Protein than to the non-mutant hPIV-4B HN Protein. This study indicated that the antigenic diversity between hPIV-4A and -4B was partly caused by deletion or creation of glycosylation sites, showing that the point mutations resulting in deletion or creation of glycosylation sites is one of the initial steps leading to the division of virus into subtypes.
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Identification of regions on the fusion Protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase Proteins to promote cell fusion.
Journal of General Virology, 1998Co-Authors: Masato Tsurudome, Morihiro Ito, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Shigeru Kusagawa, Kousuke Okamoto, Yasuhiko ItoAbstract:Using a plasmid expression system in HeLa cells, we have previously shown that the fusion (F) Protein of simian virus 41 (SV-41) induces cell fusion when coexpressed with the haemagglutinin-neuraminidase (HN) Protein of human parainfluenza virus type 2 (PIV-2), while the PIV-2 F Protein does not induce cell fusion with the SV-41 HN Protein. In the present study, we found that the PIV-2 F Protein induced extensive cell fusion with the HN Protein of mumps virus (MuV), whereas the SV-41 F Protein did not. Chimaeric analyses of the F Proteins of PIV-2 and SV-41 identified two regions (designated M1 and M2) on the PIV-2 F Protein, either of which was necessary for chimaeric F Proteins to show fusogenic activity with the MuV HN Protein. Subsequently, two additional regions (P1 and P2) were identified on the PIV-2 F Protein, both of which were necessary for chimaeric F Proteins to prevent induction of cell fusion with the SV-41 HN Protein. Consequently, it was proved that a given chimaeric F Protein, harbouring regions P1 and P2 together with either of region M1 or M2, induced cell fusion specifically with HN Proteins of PIV-2 and MuV, the same as the PIV-2 F Protein. Region M2 was located at the membrane proximal end of the PIV-2 F1 ectodomain, while regions P1, M1 and P2 clustered together in the middle of the ectodomain. These regions on the PIV-2 F Protein may be involved in a putative functional interaction with HN Proteins, which is considered to be a prerequisite for cell fusion.
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Identification of regions on the hemagglutinin-neuraminidase Protein of human parainfluenza virus type 2 important for promoting cell fusion
Virology, 1995Co-Authors: Masato Tsurudome, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Tetsuya Yuasa, Nobutada Tabata, Yasuhiko ItoAbstract:The hemagglutinin-neuraminidase (HN) and fusion (F) glycoProteins of two paramyxoviruses, human parainfluenza virus type 2 (PIV2) and simian virus 41 (SV41), were expressed in HeLa cells by transfecting with recombinant plasmid harboring each glycoProtein gene. Expressed F Proteins could not induce cell fusion by themselves, but evoked prominent cell fusion when coexpressed with homologous HN Proteins. It was also proved that PIV2 HN Protein could weakly promote SV41 F-mediated cell fusion. By analyzing the fusion-promoting function of chimeric HN Proteins of PIV2 and SV41, it was revealed that the N-terminal region (about 16% of total amino acids) of either PIV2 HN or SV41 HN Protein could define the type-specific fusion-promoting function for homologous F Protein. Analyses of additional chimeras indicated that the N-terminal region in PIV2 HN Protein (designated region I, consisting of 94 amino acids) could be reduced to a 58-amino-acid region (region I') which was located at the membrane-proximal end of the ectodomain. Furthermore, PIV2 HN Protein proved to promote cell fusion mediated by PIV4A F Protein. Unexpectedly, analyses of another set of chimeras revealed that the promoting function of PIV2 HN Protein for PIV4A F-mediated cell fusion was not merely carried by its region I but also by another region ranging from residue 148 to 209 (region II). Finally, it was indicated that regions I' (in the presumed stalk domain) and II (in the globular head) in PIV2 HN Protein might play important roles in promoting cell fusion mediated by the F Proteins.
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A cell fusion-inhibiting monoclonal antibody binds to the presumed stalk domain of the human parainfluenza type 2 virus hemagglutinin-neuraminidase Protein.
Virology, 1995Co-Authors: Tetsuya Yuasa, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Nobutada Tabata, Yasuhiko Ito, Shigeru Kusagawa, Haruo Matsumura, Masato TsurudomeAbstract:Previously, we obtained a neutralizing monoclonal antibody directed against the hemagglutinin-neuraminidase (HN) Protein of human parainfluenza type 2 virus (PIV2), which was able to prevent cell fusion without affecting the hemagglutinating and neuraminidase activities. In this study, four escape mutants of PIV2 have been obtained under pressure of the monoclonal antibody. Intriguingly, the HN Protein of each mutant proved to have two amino acid substitutions, one of which is at 83Asn or 91Lys, and another one is at 150Leu, 160Ala, or 186Met. One mutant designated F13, which has substitutions at 83Asn and 186Met in the HN Protein, could not cause cell fusion in HeLa cells despite its multiple replication, while the other mutants formed typical syncytial cells. The deduced amino acid sequence of F13 fusion (F) Protein proved to be identical to that of wild-type F Protein, and furthermore, Protein expression analyses have revealed that the low-fusion phenotype of F13 was due to its mutated HN Protein, whose antigenicity to the monoclonal antibody was abolished by the single mutation at 83Asn. These observations have suggested that the principal epitope for the monoclonal antibody resides in the presumed stalk domain of the HN Protein, which may play an important role in promoting cell fusion.