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1
Gravel, Kathryn A., and Trudy G. Morrison. "Interacting Domains of the HN and F Proteins of Newcastle Disease Virus." Journal of Virology 77, no. 20 (2003): 11040–49. http://dx.doi.org/10.1128/jvi.77.20.11040-11049.2003.
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ABSTRACT 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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McGinnes, L. W., and T. G. Morrison. "Inhibition of Receptor Binding Stabilizes Newcastle Disease Virus HN and F Protein-Containing Complexes." Journal of Virology 80, no. 6 (2006): 2894–903. http://dx.doi.org/10.1128/jvi.80.6.2894-2903.2006.
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ABSTRACT 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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McGinnes, Lori W., Kathryn Gravel, and Trudy G. Morrison. "Newcastle Disease Virus HN Protein Alters the Conformation of the F Protein at Cell Surfaces." Journal of Virology 76, no. 24 (2002): 12622–33. http://dx.doi.org/10.1128/jvi.76.24.12622-12633.2002.
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ABSTRACT 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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Tsurudome, Masato, Morihiro Ito, Junpei Ohtsuka, et al. "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 89, no. 24 (2015): 12374–87. http://dx.doi.org/10.1128/jvi.01448-15.
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ABSTRACTVirus-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.IMPORTANCEIt 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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Pantua, Homer D., Lori W. McGinnes, Mark E. Peeples, and Trudy G. Morrison. "Requirements for the Assembly and Release of Newcastle Disease Virus-Like Particles." Journal of Virology 80, no. 22 (2006): 11062–73. http://dx.doi.org/10.1128/jvi.00726-06.
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ABSTRACT Paramyxoviruses, such as Newcastle disease virus (NDV), assemble in and bud from plasma membranes of infected cells. To explore the role of each of the NDV structural proteins in virion assembly and release, virus-like particles (VLPs) released from avian cells expressing all possible combinations of the nucleoprotein (NP), membrane or matrix protein (M), an uncleaved fusion protein (F-K115Q), and hemagglutinin-neuraminidase (HN) protein were characterized for densities, protein content, and efficiencies of release. Coexpression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3 and 83.8% ± 1.1%, respectively) similar to those of authentic virions. Expression of M protein alone, but not NP, F-K115Q, or HN protein individually, resulted in efficient VLP release, and expression of all different combinations of proteins in the absence of M protein did not result in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F, and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by coimmunoprecipitation. The colocalization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M-HN and M-NP interactions are responsible for incorporation of HN and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein.
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Melanson, Vanessa R., and Ronald M. Iorio. "Addition of N-Glycans in the Stalk of the Newcastle Disease Virus HN Protein Blocks Its Interaction with the F Protein and Prevents Fusion." Journal of Virology 80, no. 2 (2006): 623–33. http://dx.doi.org/10.1128/jvi.80.2.623-633.2006.
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ABSTRACT Most paramyxovirus fusion (F) proteins require the coexpression of the homologous attachment (HN) protein to promote membrane fusion, consistent with the existence of a virus-specific interaction between the two proteins. Analysis of the fusion activities of chimeric HN proteins indicates that the stalk region of the HN spike determines its F protein specificity, and analysis of a panel of site-directed mutants indicates that the F-interactive site resides in this region. Here, we use the addition of oligosaccharides to further explore the role of the HN stalk in the interaction with F. N-glycans were individually added at several positions in the stalk to determine their effects on the activities of HN, as well as its structure. N-glycan addition at positions 69 and 77 in the stalk specifically blocks fusion and the HN-F interaction without affecting either HN structure or its other activities. N-glycans added at other positions in the stalk modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as indicated by sucrose gradient sedimentation analyses. Finally, N-glycan addition in another region of HN (residues 124 to 152), predicted by a peptide-based analysis to mediate the interaction with F, does not significantly reduce the level of fusion, arguing strongly against this site being part of the F-interactive domain in HN. Our data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.
7
Li, Jianrong, Edward Quinlan, Anne Mirza, and Ronald M. Iorio. "Mutated Form of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Interacts with the Homologous Fusion Protein despite Deficiencies in both Receptor Recognition and Fusion Promotion." Journal of Virology 78, no. 10 (2004): 5299–310. http://dx.doi.org/10.1128/jvi.78.10.5299-5310.2004.
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ABSTRACT The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segments from HN proteins derived from two different NDV strains. These findings suggest that the I175E substitution converts HN to an F-interactive form, but it is one for which receptor binding is still required for fusion promotion. The data also indicate that the integrity of the HN dimer interface is critical to its receptor recognition activity.
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Takimoto, Toru, Garry L. Taylor, Helen C. Connaris, Susan J. Crennell, and Allen Portner. "Role of the Hemagglutinin-Neuraminidase Protein in the Mechanism of Paramyxovirus-Cell Membrane Fusion." Journal of Virology 76, no. 24 (2002): 13028–33. http://dx.doi.org/10.1128/jvi.76.24.13028-13033.2002.
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ABSTRACT Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth β sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.
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Ng, D. T., S. W. Hiebert, and R. A. Lamb. "Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase." Molecular and Cellular Biology 10, no. 5 (1990): 1989–2001. http://dx.doi.org/10.1128/mcb.10.5.1989-2001.1990.
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The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.
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Ng, D. T., S. W. Hiebert, and R. A. Lamb. "Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase." Molecular and Cellular Biology 10, no. 5 (1990): 1989–2001. http://dx.doi.org/10.1128/mcb.10.5.1989.
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The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.
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Li, Jianrong, Vanessa R. Melanson, Anne M. Mirza, and Ronald M. Iorio. "Decreased Dependence on Receptor Recognition for the Fusion Promotion Activity of L289A-Mutated Newcastle Disease Virus Fusion Protein Correlates with a Monoclonal Antibody-Detected Conformational Change." Journal of Virology 79, no. 2 (2005): 1180–90. http://dx.doi.org/10.1128/jvi.79.2.1180-1190.2005.
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ABSTRACT It has been shown that the L289A-mutated Newcastle disease virus (NDV) fusion (F) protein gains the ability to promote fusion of Cos-7 cells independent of the viral hemagglutinin-neuraminidase (HN) protein and exhibits a 50% enhancement in HN-dependent fusion over wild-type (wt) F protein. Here, we show that HN-independent fusion by L289A-F is not exhibited in BHK cells or in several other cell lines. However, similar to the results in Cos-7 cells, the mutated protein plus HN does promote 50 to 70% more fusion above wt levels in all of the cell lines tested. L289A-F protein exhibits the same specificity as the wt F protein for the homologous HN protein, as well as NDV-human parainfluenza virus 3 HN chimeras. The mutated F protein promotes fusion more effectively than the wt when it is coexpressed with either the chimeras or HN proteins deficient in receptor recognition activity. In addition, its fusogenic activity is significantly more resistant to removal of sialic acid on target cells. These findings are consistent with the demonstration that L289A-F interacts more efficiently with wt and mutated HN proteins than does wt F by a cell surface coimmunoprecipitation assay. Taken together, these findings indicate that L289A-F promotes fusion by a mechanism analogous to that of the wt protein with respect to the HN-F interaction but is less dependent on the attachment activity of HN. The phenotype of the mutated F protein correlates with a conformational change in the protein detectable by two different monoclonal antibodies. This conformational change may reflect a destabilization of F structure induced by the L289A substitution, which may in turn indicate a lower energy requirement for fusion activation.
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Dutch, Rebecca Ellis, Sangeeta Bagai Joshi, and Robert A. Lamb. "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 72, no. 10 (1998): 7745–53. http://dx.doi.org/10.1128/jvi.72.10.7745-7753.1998.
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ABSTRACT 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.
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Iorio, Ronald M., Gisela M. Field, Jennifer M. Sauvron, et al. "Structural and Functional Relationship between the Receptor Recognition and Neuraminidase Activities of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein: Receptor Recognition Is Dependent on Neuraminidase Activity." Journal of Virology 75, no. 4 (2001): 1918–27. http://dx.doi.org/10.1128/jvi.75.4.1918-1927.2001.
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ABSTRACT The terminal globular domain of the paramyxovirus hemagglutinin-neuraminidase (HN) glycoprotein spike has a number of conserved residues that are predicted to form its neuraminidase (NA) active site, by analogy to the influenza virus neuraminidase protein. We have performed a site-directed mutational analysis of the role of these residues in the functional activity of the Newcastle disease virus (NDV) HN protein. Substitutions for several of these residues result in a protein lacking both detectable NA and receptor recognition activity. Contribution of NA activity, either exogenously or by coexpression with another HN protein, partially rescues the receptor recognition activity of these proteins, indicating that the receptor recognition deficiencies of the mutated HN proteins result from their lack of detectable NA activity. In addition to providing support for the homology-based predictions for the structure of HN, these findings argue that (i) the HN residues that mediate its NA activity are not critical to its attachment function and (ii) NA activity is required for the protein to mediate binding to receptors.
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Takimoto, Toru, Tatiana Bousse, Elizabeth C. Coronel, Ruth Ann Scroggs, and Allen Portner. "Cytoplasmic Domain of Sendai Virus HN Protein Contains a Specific Sequence Required for Its Incorporation into Virions." Journal of Virology 72, no. 12 (1998): 9747–54. http://dx.doi.org/10.1128/jvi.72.12.9747-9754.1998.
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ABSTRACT In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.
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Zhai, Dayong, Frederic Luciano, Xiuwen Zhu, Bin Guo, Arnold C. Satterthwait, and John C. Reed. "Humanin Binds and Nullifies Bid Activity by Blocking Its Activation of Bax and Bak." Journal of Biological Chemistry 280, no. 16 (2005): 15815–24. http://dx.doi.org/10.1074/jbc.m411902200.
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Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified Bid and tBidin vitroand blocks tBid-induced release of cytochromecand SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover, HN peptide also retained protective activity onbax–/–mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this endogenous peptide.
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Lu, Xiaolong, Tiansong Zhan, Kaituo Liu, et al. "Biological Significance of Dual Mutations A494D and E495K of the Genotype III Newcastle Disease Virus Hemagglutinin-Neuraminidase In Vitro and In Vivo." Viruses 14, no. 11 (2022): 2338. http://dx.doi.org/10.3390/v14112338.
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As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.
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Krishnan, Anuja, Santosh K. Verma, Prashant Mani, Rahul Gupta, Suman Kundu, and Debi P. Sarkar. "A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion." Journal of Virology 83, no. 4 (2008): 1727–41. http://dx.doi.org/10.1128/jvi.02026-08.
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ABSTRACT Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.
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Huang, Zhuhui, Aruna Panda, Subbiah Elankumaran, Dhanasekaran Govindarajan, Daniel D. Rockemann, and Siba K. Samal. "The Hemagglutinin-Neuraminidase Protein of Newcastle Disease Virus Determines Tropism and Virulence." Journal of Virology 78, no. 8 (2004): 4176–84. http://dx.doi.org/10.1128/jvi.78.8.4176-4184.2004.
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ABSTRACT The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.
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Connolly, Sarah A., George P. Leser, Theodore S. Jardetzky, and Robert A. Lamb. "Bimolecular Complementation of Paramyxovirus Fusion and Hemagglutinin-Neuraminidase Proteins Enhances Fusion: Implications for the Mechanism of Fusion Triggering." Journal of Virology 83, no. 21 (2009): 10857–68. http://dx.doi.org/10.1128/jvi.01191-09.
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ABSTRACT For paramyxoviruses, entry requires a receptor-binding protein (hemagglutinin-neuraminidase [HN], H, or G) and a fusion protein (F). Like other class I viral fusion proteins, F is expressed as a prefusion metastable protein that undergoes a refolding event to induce fusion. HN binding to its receptor triggers F refolding by an unknown mechanism. HN may serve as a clamp that stabilizes F in its prefusion state until HN binds the target cell (the “clamp model”). Alternatively, HN itself may undergo a conformational change after receptor binding that destabilizes F and causes F to trigger (the “provocateur model”). To examine F-HN interactions by bimolecular fluorescence complementation (BiFC), the cytoplasmic tails of parainfluenza virus 5 (PIV5) F and HN were fused to complementary fragments of yellow fluorescent protein (YFP). Coexpression of the BiFC constructs resulted in fluorescence; however, coexpression with unrelated BiFC constructs also produced fluorescence. The affinity of the two halves of YFP presumably superseded the F-HN interaction. Unexpectedly, coexpression of the BiFC F and HN constructs greatly enhanced fusion in multiple cell types. We hypothesize that the increase in fusion occurs because the BiFC tags bring F and HN together more frequently than occurs in a wild-type (wt) scenario. This implies that normally much of wt F is not associated with wt HN, in conflict with the clamp model for activation. Correspondingly, we show that wt PIV5 fusion occurs in an HN concentration-dependent manner. Also inconsistent with the clamp model are the findings that BiFC F does not adopt a postfusion conformation when expressed in the absence of HN and that HN coexpression does not provide resistance to the heat-induced triggering of F. In support of a provocateur model of F activation, we demonstrate by analysis of the morphology of soluble F trimers that the hyperfusogenic mutation S443P has a destabilizing effect on F.
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Wilson, C., R. Gilmore, and T. Morrison. "Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus." Molecular and Cellular Biology 7, no. 4 (1987): 1386–92. http://dx.doi.org/10.1128/mcb.7.4.1386-1392.1987.
Full textAbstract:
The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses is likely in the unusual class of glycoproteins with the amino terminus cytoplasmic and the carboxy terminus lumenal or external to the cell. The properties of the membrane insertion of the HN protein of Newcastle disease virus, a prototype paramyxovirus, were explored in wheat germ extracts containing microsomal membranes. HN protein was inserted into membranes cotranslationally, resulting in a glycosylated protein completely resistant to trypsin and proteinase K digestion. No detectable posttranslation insertion occurred. Insertion required signal recognition particle. Signal recognition particle in the absence of membranes inhibited HN protein synthesis. Comparisons of the trypsin digestion products of the HN protein made in the cell-free system with newly synthesized HN protein from infected cells showed that the cell-free product was in a conformation different from that of the pulse-labeled protein in infected cells. First, trypsin digestion of intact membranes from infected cells reduced the size of the 74,000-dalton HN protein by approximately 1,000 daltons, whereas trypsin digestion of HN protein made in the cell-free system had no effect on the size of the protein. Second, trypsin digestion of Triton X-100-permeabilized membranes isolated from infected cells resulted in a 67,000-dalton trypsin resistant HN protein fragment. A trypsin-resistant core of comparable size was not present in the digestion products of in-vitro-synthesized HN protein. Evidence is presented that the newly synthesized HN protein in infected cels contain intramolecular disulfide bonds not present in the cell-free product.
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Wilson, C., R. Gilmore, and T. Morrison. "Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus." Molecular and Cellular Biology 7, no. 4 (1987): 1386–92. http://dx.doi.org/10.1128/mcb.7.4.1386.
Full textAbstract:
The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses is likely in the unusual class of glycoproteins with the amino terminus cytoplasmic and the carboxy terminus lumenal or external to the cell. The properties of the membrane insertion of the HN protein of Newcastle disease virus, a prototype paramyxovirus, were explored in wheat germ extracts containing microsomal membranes. HN protein was inserted into membranes cotranslationally, resulting in a glycosylated protein completely resistant to trypsin and proteinase K digestion. No detectable posttranslation insertion occurred. Insertion required signal recognition particle. Signal recognition particle in the absence of membranes inhibited HN protein synthesis. Comparisons of the trypsin digestion products of the HN protein made in the cell-free system with newly synthesized HN protein from infected cells showed that the cell-free product was in a conformation different from that of the pulse-labeled protein in infected cells. First, trypsin digestion of intact membranes from infected cells reduced the size of the 74,000-dalton HN protein by approximately 1,000 daltons, whereas trypsin digestion of HN protein made in the cell-free system had no effect on the size of the protein. Second, trypsin digestion of Triton X-100-permeabilized membranes isolated from infected cells resulted in a 67,000-dalton trypsin resistant HN protein fragment. A trypsin-resistant core of comparable size was not present in the digestion products of in-vitro-synthesized HN protein. Evidence is presented that the newly synthesized HN protein in infected cels contain intramolecular disulfide bonds not present in the cell-free product.
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Ren, Guijie, Yunlong Zhuang, Keli Tian, et al. "Acidic amino acids increase fusion activity in the specific fusion domain of Newcastle disease virus fusion protein." Canadian Journal of Microbiology 59, no. 9 (2013): 641–44. http://dx.doi.org/10.1139/cjm-2013-0133.
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To explore the effects of amino acids Gln and Asn within the specific fusion domain of fusion (F) protein on the specific membrane fusion in Newcastle disease virus (NDV), the mutants Q204E–Q205E and N245D were constructed in the specific fusion domain of F protein. The mutant genes were co-expressed with homologous or heterologous hemagglutinin–neuraminidase (HN) in BHK21 cells. Cell fusion functions of mutants were analyzed with Giemsa staining and reporter gene methods. Cell surface expression efficiency was analyzed with immunofluorescence assay and fluorescence-activated cell sorter analysis. Co-immunoprecipitation was performed to analyze the interaction of mutant F proteins with the homotypic HN protein. Both Q204E–Q205E and N245D mutations caused increased cell–cell fusion activity when they were co-expressed with homotypic HN protein. The mutant F proteins had slight changes in cell surface expression compared with that of wild-type F protein. The interactions of Q204E–Q205E or N245D with their homotypic HN increased significantly (P < 0.01) compared with the wild-type F protein. Neither Q204–Q205E nor N245D caused cell fusion in the presence of heterologous HN protein. Our data suggested that the residues Q204, Q205, and N245 play a critical role in the regulation of cell fusion. They may decrease the interaction of wild-type NDV F and NDV HN to suppress the fusion activity for survival of the infected host, which may enable a persistent virus infection and long-term virus reproduction and spread.
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Leser, G. P., K. J. Ector, and R. A. Lamb. "The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway." Molecular Biology of the Cell 7, no. 1 (1996): 155–72. http://dx.doi.org/10.1091/mbc.7.1.155.
Full textAbstract:
The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.
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Cheng, Boxing, Fei Liu, Qiaosheng Guo, et al. "Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia." PeerJ 7 (September 30, 2019): e7716. http://dx.doi.org/10.7717/peerj.7716.
Full textAbstract:
Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leech Hirudo nipponia was obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 from Hirudo medicinalis with an e-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (Hir and M-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.
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Jain, Surbhi, Lori W. McGinnes, and Trudy G. Morrison. "Role of Thiol/Disulfide Exchange in Newcastle Disease Virus Entry." Journal of Virology 83, no. 1 (2008): 241–49. http://dx.doi.org/10.1128/jvi.01407-08.
Full textAbstract:
ABSTRACT Newcastle disease virus (NDV) entry into host cells is mediated by the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. We previously showed that production of free thiols in F protein is required for membrane fusion directed by F protein (S. Jain et al., J. Virol. 81:2328-2339, 2007). In the present study we evaluated the oxidation state of F protein in virions and virus-like particles and its relationship to activation of F protein by HN protein, F protein conformational intermediates, and virus-cell fusion. F protein, in particles, does not have free thiols, but free thiols were produced upon binding of particles to target cells. Free thiols were produced at 16°C in F protein in virions bound to the target cells. They also appeared in different fusion defective mutant F proteins. Free thiols were produced in the presence of mutant HN proteins that are defective in F protein activation but are attachment competent. These results suggest that free thiols appear prior to any of the proposed major conformational changes in F protein which accompany fusion activation. These results also indicate that HN protein binding to its receptor likely facilitates the interaction between F protein and host cell isomerases, leading to reduction of disulfide bonds in F protein. Taken together, these results show that free thiols are produced in F protein at a very early stage during the onset of fusion and that the production of free thiols is required for fusion in addition to activation by HN protein.
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Stone-Hulslander, Judith, and Trudy G. Morrison. "Mutational Analysis of Heptad Repeats in the Membrane-Proximal Region of Newcastle Disease Virus HN Protein." Journal of Virology 73, no. 5 (1999): 3630–37. http://dx.doi.org/10.1128/jvi.73.5.3630-3637.1999.
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ABSTRACT For most paramyxoviruses, syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (X. Hu et al., J. Virol. 66:1528–1534, 1992). The stalk region of the Newcastle disease virus (NDV) HN protein has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (R. Buckland et al., J. Gen. Virol. 73:1703–1707, 1992; T. Sergel-Germano et al., J. Virol. 68:7654–7658, 1994; J. Reitter et al., J. Virol. 69:5995–6004, 1995). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic a-position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein activity and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.
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Sergel, Theresa A., Lori W. McGinnes, and Trudy G. Morrison. "A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion." Journal of Virology 74, no. 11 (2000): 5101–7. http://dx.doi.org/10.1128/jvi.74.11.5101-5107.2000.
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ABSTRACT 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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McGinnes, Lori W., Homer Pantua, Jason P. Laliberte, Kathryn A. Gravel, Surbhi Jain, and Trudy G. Morrison. "Assembly and Biological and Immunological Properties of Newcastle Disease Virus-Like Particles." Journal of Virology 84, no. 9 (2010): 4513–23. http://dx.doi.org/10.1128/jvi.01931-09.
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ABSTRACT Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.
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Porotto, Matteo, Matthew Murrell, Olga Greengard, and Anne Moscona. "Triggering of Human Parainfluenza Virus 3 Fusion Protein (F) by the Hemagglutinin-Neuraminidase (HN) Protein: an HN Mutation Diminishes the Rate of F Activation and Fusion." Journal of Virology 77, no. 6 (2003): 3647–54. http://dx.doi.org/10.1128/jvi.77.6.3647-3654.2003.
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ABSTRACT For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4°C are transferred to 22°C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22°C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN′s F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.
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Luciano, Frederic, Dayong Zhai, Xiuwen Zhu, et al. "Cytoprotective Peptide Humanin Binds and Inhibits Proapoptotic Bcl-2/Bax Family Protein BimEL." Journal of Biological Chemistry 280, no. 16 (2005): 15825–35. http://dx.doi.org/10.1074/jbc.m413062200.
Full textAbstract:
Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Usingin vitroprotein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochromecfrom mitochondria isolated frombax–/–cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.
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Kobayashi, Masanori, Akihiro Iida, Yasuji Ueda, and Mamoru Hasegawa. "Pseudotyped Lentivirus Vectors Derived from Simian Immunodeficiency Virus SIVagm with Envelope Glycoproteins from Paramyxovirus." Journal of Virology 77, no. 4 (2003): 2607–14. http://dx.doi.org/10.1128/jvi.77.4.2607-2614.2003.
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ABSTRACT We describe the development of novel lentivirus vectors based on simian immunodeficiency virus from African green monkey (SIVagm) pseudotyped with Sendai virus (SeV) envelope glycoproteins. SeV fusion (F) and hemagglutinin-neuraminidase (HN) proteins were successfully incorporated into the SIVagm-based vector by truncation of the cytoplasmic tail of the F protein and by addition of the cytoplasmic tail of SIVagm transmembrane envelope protein to the N terminus of the HN protein. As with the vesicular stomatitis virus G glycoprotein-pseudotyped vector, the mutant SeV F- and HN-pseudotyped SIVagm vector was able to transduce various types of animal and human cell lines. Furthermore, the vector was able to transduce an enhanced green fluorescent protein reporter gene into polarized epithelial cells of rat trachea from the apical and basolateral sides. Therefore, SeV F- and HN-pseudotyped SIVagm vectors have considerable potential for effective use in gene therapy for various therapies, including respiratory diseases.
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Jain, Surbhi, Lori W. McGinnes, and Trudy G. Morrison. "Thiol/Disulfide Exchange Is Required for Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein." Journal of Virology 81, no. 5 (2006): 2328–39. http://dx.doi.org/10.1128/jvi.01940-06.
Full textAbstract:
ABSTRACT 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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Wilson, C., T. Connolly, T. Morrison, and R. Gilmore. "Integration of membrane proteins into the endoplasmic reticulum requires GTP." Journal of Cell Biology 107, no. 1 (1988): 69–77. http://dx.doi.org/10.1083/jcb.107.1.69.
Full textAbstract:
We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.
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Feng, Y., B. Press, and A. Wandinger-Ness. "Rab 7: an important regulator of late endocytic membrane traffic." Journal of Cell Biology 131, no. 6 (1995): 1435–52. http://dx.doi.org/10.1083/jcb.131.6.1435.
Full textAbstract:
Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.
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Mahon, Paul J., Anne M. Mirza, Thomas A. Musich, and Ronald M. Iorio. "Engineered Intermonomeric Disulfide Bonds in the Globular Domain of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein: Implications for the Mechanism of Fusion Promotion." Journal of Virology 82, no. 21 (2008): 10386–96. http://dx.doi.org/10.1128/jvi.00581-08.
Full textAbstract:
ABSTRACT The promotion of membrane fusion by Newcastle disease virus (NDV) requires an interaction between the viral hemagglutinin-neuraminidase (HN) and fusion (F) proteins, although the mechanism by which this interaction regulates fusion is not clear. The NDV HN protein exists as a tetramer composed of a pair of dimers. Based on X-ray crystallographic studies of the NDV HN globular domain (S. Crennell et al., Nat. Struct. Biol. 7:1068-1074, 2000), it was proposed that the protein undergoes a significant conformational change from an initial structure having minimal intermonomeric contacts to a structure with a much more extensive dimer interface. This conformational change was predicted to be integral to fusion promotion with the minimal interface form required to maintain F in its prefusion state until HN binds receptors. However, no evidence for such a conformational change exists for any other paramyxovirus attachment protein. To test the NDV model, we have engineered a pair of intermonomeric disulfide bonds across the dimer interface in the globular domain of an otherwise non-disulfide-linked NDV HN protein by the introduction of cysteine substitutions for residues T216 and D230. The disulfide-linked dimer is formed both intracellularly and in the absence of receptor binding and is efficiently expressed at the cell surface. The disulfide bonds preclude formation of the minimal interface form of the protein and yet enhance both receptor-binding activity at 37°C and fusion promotion. These results confirm that neither the minimal interface form of HN nor the proposed drastic conformational change in the protein is required for fusion.
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Melanson, Vanessa R., and Ronald M. Iorio. "Amino Acid Substitutions in the F-Specific Domain in the Stalk of the Newcastle Disease Virus HN Protein Modulate Fusion and Interfere with Its Interaction with the F Protein." Journal of Virology 78, no. 23 (2004): 13053–61. http://dx.doi.org/10.1128/jvi.78.23.13053-13061.2004.
Full textAbstract:
ABSTRACT The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus mediates attachment to sialic acid receptors, as well as cleavage of the same moiety. HN also interacts with the other viral glycoprotein, the fusion (F) protein, to promote membrane fusion. The ectodomain of the HN spike consists of a stalk and a terminal globular head. The most conserved part of the stalk consists of two heptad repeats separated by a nonhelical intervening region (residues 89 to 95). Several amino acid substitutions for a completely conserved proline residue in this region not only impair fusion and the HN-F interaction but also decrease neuraminidase activity in the globular domain, suggesting that the substitutions may alter HN structure. Substitutions for L94 also interfere with fusion and the HN-F interaction but have no significant effect on any other HN function. Amino acid substitutions at other positions in the intervening region also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the intervening region is critical to the role of HN in the promotion of fusion and may be directly involved in its interaction with the homologous F protein.
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Earl, R. T., E. E. Billett, I. M. Hunneyball, and R. J. Mayer. "Sendai-viral HN and F glycoproteins as probes of plasma-membrane protein catabolism in HTC cells. Studies with fusogenic reconstituted Sendai-viral envelopes." Biochemical Journal 241, no. 3 (1987): 801–7. http://dx.doi.org/10.1042/bj2410801.
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Reconstituted Sendai-viral envelopes (RSVE) were produced by the method of Vainstein, Hershkovitz, Israel & Loyter [(1984) Biochim. Biophys. Acta 773, 181-188]. RSVE are fusogenic unilamellar vesicles containing two transmembrane glycoproteins: the HN (haemagglutinin-neuraminidase) protein and the F (fusion) factor. The fate of the viral proteins after fusion-mediated transplantation of RSVE into hepatoma (HTC) cell plasma membranes was studied to probe plasma-membrane protein degradation. Both protein species are degraded at similar, relatively slow, rates (t1/2 = 67 h) in HTC cells fused with RSVE in suspension. Even slower degradation rates for HN and F proteins (t1/2 = 93 h) were measured when RSVE were fused with HTC cells in monolayer. Lysosomal degradation of the transplanted viral proteins is strongly implicated by the finding that degradation of HN and F proteins is sensitive to inhibition by 10 mM-NH4Cl (81%) and by 50 micrograms of leupeptin/ml (70%).
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Le, Van Truong, Hong Phuoc Man, and Van Quyen Dong. "Expression of haemagglutinin-neuraminidase of newcastle virus genotype VIII in insect cell Sf9." Academia Journal of Biology 46, no. 1 (2024): 79–86. http://dx.doi.org/10.15625/2615-9023/18907.
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The hemagglutinin-neuraminidase (HN) protein is the surface glycoprotein of Newcastle disease virus (NDV) which is involved in the process of virus entry into host cells. HN exhibits the capability to induce agglutination of chicken red blood cells, thereby contributing to the viral infection of the host. As a consequence, HN is a potential antigen for the production of subunit vaccines for protecting poultry from NDV infections. In this study, the hn gene coding for the HN antigen of NDV genotype VIII was artificially synthesized, ligated into donor vector pFastBacHT A and integrated into the baculovirus genome for the purpose of HN expression in Spodoptera frugiperda 9 (Sf9) cells. The recombinant HN protein was successfully expressed in Sf9 cells in the SF900 III medium. The expressed recombinant HN protein, with a size of approximately 63 kDa, exhibited hemagglutinin activity (HA) of 4log2 when interacting with chicken erythrocyte fluid. The findings of this study lay the foundation for subsequent research endeavors aimed at the development of Newcastle disease virus (NDV) vaccines tailored for poultry in Vietnam.
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Ng, D. T., R. E. Randall, and R. A. Lamb. "Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface." Journal of Cell Biology 109, no. 6 (1989): 3273–89. http://dx.doi.org/10.1083/jcb.109.6.3273.
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The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is a type II integral membrane protein that is expressed at the infected cell surface. The intracellular assembly and transport of HN in CV1 cells was examined using conformation-specific HN mAbs and sucrose density sedimentation analysis. HN was found to oligomerize with a t1/2 of 25-30 min and these data suggest the oligomer is a tetramer consisting primarily of two noncovalently associated disulfide-linked dimers. As HN oligomers could be found that were sensitive to endoglycosidase H digestion and oligomers formed in the presence of the ER to the Golgi complex transport inhibitor, carbonylcyanide m-chlorophenylhydrazone (CCCP), these data are consistent with HN oligomerization occurring in the ER. Unfolded or immature HN molecules that could not be recognized by conformation-specific antibodies were found to specifically associate with the resident ER protein GRP78-BiP. Immunoprecipitation of BiP-HN complexes with an immunoglobulin heavy-chain binding protein (BiP) antibody indicated that newly synthesized HN associated and dissociated from GRP78-BiP (t1/2 20-25 min) in an inverse correlation with the gain in reactivity with a HN conformation-specific antibody, suggesting that the transient association of GRP78-BiP with immature HN is part of the normal HN maturation pathway. After pulse-labeling of HN in infected cells, it was found that HN is rapidly turned over in cells (t1/2 2-2.5 h). This led to the finding that the vast majority of HN expressed at the cell surface, rather than being incorporated into budding virions, is internalized and degraded after localization to endocytic vesicles and lysosomes.
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Li, Zhiying, Bolin Li, Jiansheng Lu, et al. "Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F." Toxins 15, no. 3 (2023): 200. http://dx.doi.org/10.3390/toxins15030200.
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Botulinum neurotoxins (BoNTs) can cause nerve paralysis syndrome in mammals and other vertebrates. BoNTs are the most toxic biotoxins known and are classified as Class A biological warfare agents. BoNTs are mainly divided into seven serotypes A-G and new neurotoxins BoNT/H and BoNT/X, which have similar functions. BoNT proteins are 150 kDa polypeptide consisting of two chains and three domains: the light chain (L, catalytic domain, 50 kDa) and the heavy chain (H, 100 kDa), which can be divided into an N-terminal membrane translocation domain (HN, 50 kDa) and a C-terminal receptor binding domain (Hc, 50 kDa). In current study, we explored the immunoprotective efficacy of each functional molecule of BoNT/F and the biological characteristics of the light chain-heavy N-terminal domain (FL-HN). The two structure forms of FL-HN (i.e., FL-HN-SC: single chain FL-HN and FL-HN-DC: di-chain FL-HN) were developed and identified. FL-HN-SC could cleave the vesicle associated membrane protein 2 (VAMP2) substrate protein in vitro as FL-HN-DC or FL. While only FL-HN-DC had neurotoxicity and could enter neuro-2a cells to cleave VAMP2. Our results showed that the FL-HN-SC had a better immune protection effect than the Hc of BoNT/F (FHc), which indicated that L-HN-SC, as an antigen, provided the strongest protective effects against BoNT/F among all the tested functional molecules. Further in-depth research on the different molecular forms of FL-HN suggested that there were some important antibody epitopes at the L-HN junction of BoNT/F. Thus, FL-HN-SC could be used as a subunit vaccine to replace the FHc subunit vaccine and/or toxoid vaccine, and to develop antibody immune molecules targeting L and HN domains rather than the FHc domain. FL-HN-DC could be used as a new functional molecule to evaluate and explore the structure and activity of toxin molecules. Further exploration of the biological activity and molecular mechanism of the functional FL-HN or BoNT/F is warranted.
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Bousse, Tatiana, and Toru Takimoto. "Mutation at Residue 523 Creates a Second Receptor Binding Site on Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Protein." Journal of Virology 80, no. 18 (2006): 9009–16. http://dx.doi.org/10.1128/jvi.00969-06.
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ABSTRACT The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein mediating hemagglutination (HA), neuraminidase (NA), and fusion promotion activities. It has been a matter of debate whether HN contains combined or separate sites for HA and NA activities. To clear the issue, we determined the presence of the second binding site on human parainfluenza virus (hPIV) type 1, 2, and 3 and Sendai virus (SeV) HN proteins. Results of virus elution from erythrocytes at an elevated temperature and HA inhibition by NA inhibitor BCX-2798 suggest that all hPIVs bind to the receptor only through the NA catalytic site, while SeV HN has an additional receptor binding site. Comparison of SeV and hPIV1 HN sequences revealed two amino acid differences at residues 521 and 523 in the region close to the second binding site identified in Newcastle disease virus HN. We mutated hPIV1 HN at position 523 from Asn to the residue of SeV HN, Asp, and rescued a recombinant SeV that carries the mutated hPIV1 HN by a reverse genetics system. The hPIV1 HN with Asp at position 523 hemagglutinated in the presence of BCX-2798, suggesting that the amino acid difference at position 523 is critical for the formation of a second binding site. Creation of the second binding site on hPIV1 HN, however, did not significantly affect the growth or fusion activity of the recombinant virus. Our study indicates that the presence and requirement of a second binding site vary among paramyxoviruses.
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Otani, Kanako, Ryusuke Kimura, Norika Nagasawa, et al. "Phylogenomic Analyses of the Hemagglutinin-Neuraminidase (HN) Gene in Human Parainfluenza Virus Type 4 Isolates in Japan." Microorganisms 13, no. 2 (2025): 384. https://doi.org/10.3390/microorganisms13020384.
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To better understand the phylogenomics of the hemagglutinin-neuraminidase (HN) gene and HN protein in human parainfluenza virus type 4 (HPIV4), we performed phylogenomic analyses using various bioinformatics methods. The main bioinformatics analyses included a time-scaled phylogeny, genetic distance assessments, and three-dimensional (3D) structure mapping of the HN protein with conformational epitope and selective pressure analyses. The time-scaled phylogenetic tree indicated that the most recent common ancestor of the HN gene emerged approximately 100 years ago. Additionally, the tree revealed two distinct clusters corresponding to HPIV4a and HPIV4b. The divergence times for the most recent common ancestors of the HN gene in HPIV4a and HPIV4b strains were estimated to be around 1993 and 1986, respectively. The evolutionary rates of the gene varied significantly between clusters, ranging from approximately 1.2 × 10−3 to 8.7 × 10−4 substitutions per site per year. Genetic distances within each cluster were relatively short (less than 0.04). Phylodynamic analyses demonstrated an increase in the genome population size around the year 2000. Structural analyses revealed that the active sites of the HN protein were located at the protein’s head. Furthermore, the most conformational epitopes were located in adjacent active sites of the protein. These results suggested that reinfection may be unlikely to occur in the case of most HPIV4. Together, the HN gene and protein of HPIV4 strains isolated in Japan have undergone unique evolutionary changes. In addition, antibodies targeting the conformational epitopes of the HPIV4 HN protein may contribute to protection against the virus.
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Ma, Dongyun, Honghuan Gao, Chenyang Du, et al. "Transcriptomic and Metabolomics Analysis of Different Endosperm Region under Nitrogen Treatments." International Journal of Molecular Sciences 20, no. 17 (2019): 4212. http://dx.doi.org/10.3390/ijms20174212.
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Storage protein distribution in wheat-grain endosperm is heterogeneous, but the underlying molecular mechanism remains unclear. Two parts of the endosperm region, the innermost endosperm (IE) region and the remaining endosperm (RE) region, grown under low nitrogen (LN) and high nitrogen (HN) treatments were used to perform metabolomic and transcriptomic analysis. We identified 533 and 503 differentially expressed genes (DEGs) with at least a two-fold expression change (p < 0.05) between IE and RE, among which 81 and 78 transcripts under LN and HN, respectively, related to carbon and nitrogen metabolism, and encoded transcription factors or proteins involved in post-translational modification (PTM). The significantly differentially abundant metabolites between IE and RE were mainly amino acids, N-compounds, carbohydrates, and nucleic acids. More upregulated transcripts and metabolites were identified in RE than IE under HN conditions, indicating that HN activates metabolism in the endosperm periphery. In addition to carbon and nitrogen metabolism, transcription factors and protein PTMs, such as phosphorylation and acetylation, might determine the protein heterogeneous distribution between IE and RE and its response to nitrogen fertilizer supply.
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Parks, G. D., J. D. Hull, and R. A. Lamb. "Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation." Journal of Cell Biology 109, no. 5 (1989): 2023–32. http://dx.doi.org/10.1083/jcb.109.5.2023.
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The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.
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Adu-Gyamfi, Emmanuel, Lori S. Kim, Theodore S. Jardetzky, and Robert A. Lamb. "Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity." Journal of Virology 90, no. 17 (2016): 7778–88. http://dx.doi.org/10.1128/jvi.00896-16.
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ABSTRACTParamyxoviridaeconsist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering.IMPORTANCEParamyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.
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Bousse, Tatiana L., Garry Taylor, Sateesh Krishnamurthy, Allen Portner, Siba K. Samal, and Toru Takimoto. "Biological Significance of the Second Receptor Binding Site of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein." Journal of Virology 78, no. 23 (2004): 13351–55. http://dx.doi.org/10.1128/jvi.78.23.13351-13355.2004.
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ABSTRACT The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-α(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.
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Tolentino, Jarel Elgin M., Jorge Gil C. Angeles, Remil L. Galay, and Dennis V. Umali. "Reverse transcription PCR-based detection of matrix and hemagglutinin-neuraminidase genes among Avian orthoavulavirus 1 clinical isolates in the Philippines, 1991-2017." Research Journal of Chemistry and Environment 25, no. 9 (2021): 89–95. http://dx.doi.org/10.25303/259rjce8995.
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Newcastle disease (ND) is a highly infectious disease that affects devastatingly the avian population worldwide. It is caused by Avian orthoavulavirus 1 (AOAV-1), or better known as Newcastle disease virus belonging to phylum Negarnaviricota, class Monjiviricetes, order Mononegavirales and family Paramyxoviridae. This virus consists of six principal structural proteins namely: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large protein RNA dependent RNA polymerase (L).. The present study aimed to molecularly detect the M and HN gene segments of the AOAV-1 field isolates from clinical cases in the Philippines from 1991 through 2017. RT-PCR amplification and sequence analyses using primers NDV-For4359 and NDV-Rev4788 which anneal to the matrix gene and primers NDV-For6369 and NDV-Rev6598 targeting the HN genes, identified all isolates to be AOAV-1. Determining the different genes of the virus would greatly help scientists and researchers to accurately identify the viral isolates in order to improve epidemiological studies and surveillance of the disease in the country.
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Ito, Morihiro, Machiko Nishio, Mitsuo Kawano, Hiroshi Komada, Yasuhiko Ito, and Masato Tsurudome. "Effects of multiple amino acids of the parainfluenza virus 5 fusion protein on its haemagglutinin–neuraminidase-independent fusion activity." Journal of General Virology 90, no. 2 (2009): 405–13. http://dx.doi.org/10.1099/vir.0.006437-0.
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The fusion (F) protein of parainfluenza virus 5 (PIV-5) strain W3A is able to induce cell fusion when it is expressed alone in baby hamster kidney cells, whilst the F protein of PIV-5 strain WR induces cell fusion only when co-expressed with the haemagglutinin–neuraminidase (HN) protein. It has been shown previously that when Leu-22 of the WR F protein is replaced with the W3A F counterpart (Pro-22), the resulting mutant L22P exhibits HN-independent fusion activity. Furthermore, previous chimeric analysis between L22P and the F protein of PIV-5 strain T1 has suggested that Glu-132 also contributes to the HN-independent fusion activity of L22P. It was shown here that substitution of Glu-132 of L22P with various amino acids including the T1 F protein counterpart (Lys-132) resulted in a reduction in fusion activity, whereas substitution with Asp was the exception in being tolerated. Interestingly, reduced fusion activity of an L22P mutant that harboured the E132K substitution could be restored by an additional D416K substitution but not by a D416E mutation, suggesting that the presence of the same charge at positions 132 and 416 is important for the HN-independent fusion activity. In contrast, substitution of Leu-22 of the WR F protein with various amino acids except those with aliphatic side chains resulted in acquisition of fusion activity, suggesting that the HN dependence of the WR F protein in the induction of cell fusion is attributable to the hydrophobicity of Leu-22. These results indicate that at least three amino acids are involved in the HN-independent fusion activity of the PIV-5 F protein.
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Porotto, Matteo, Matthew Murrell, Olga Greengard, Lynne Doctor, and Anne Moscona. "Influence of the Human Parainfluenza Virus 3 Attachment Protein's Neuraminidase Activity on Its Capacity To Activate the Fusion Protein." Journal of Virology 79, no. 4 (2005): 2383–92. http://dx.doi.org/10.1128/jvi.79.4.2383-2392.2005.
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ABSTRACT In order to examine functions of the hemagglutinin-neuraminidase (HN) protein that quantitatively influence fusion promotion, human parainfluenza virus 3 (HPIV3) variants with alterations in HN were studied. The variant HNs have mutations that affect either receptor binding avidity, neuraminidase activity, or fusion protein (F) activation. Neuraminidase activity was regulated by manipulation of temperature and pH. F activation was assessed by quantitating the irreversible binding of target erythrocytes (RBC) to HN/F-coexpressing cells in the presence of 4-GU-DANA (zanamivir) to release target cells bound only by HN-receptor interactions; the remaining, irreversibly bound target cells are retained via the fusion protein. In cells coexpressing wild-type (wt) or variant HNs with wt F, the fusion promotion capacity of HN was distinguished from target cell binding by measuring changes with time in the amounts of target RBC that were (i) reversibly bound by HN-receptor interaction (released only upon the addition of 4-GU-DANA), (ii) released by HN′s neuraminidase, and (iii) irreversibly bound by F-insertion or fusion (F triggered). For wt HN, lowering the pH (to approach the optimum for HPIV3 neuraminidase) decreased F triggering via release of HN from its receptor. An HN variant with increased receptor binding avidity had F-triggering efficiency like that of wt HN at pH 8.0, but this efficiency was not decreased by lowering the pH to 5.7, which suggested that the variant HN′s higher receptor binding activity counterbalanced the receptor dissociation promoted by increased neuraminidase activity. To dissect the specific contribution of neuraminidase to triggering, two variant HNs that are triggering-defective due to a mutation in the HN stalk were evaluated. One of these variants has, in addition, a mutation in the globular head that renders it neuraminidase dead, while the HN with the stalk mutation alone has 30% of wt neuraminidase. While the variant without neuraminidase activity triggered F effectively at 37°C irrespective of pH, the variant possessing effective neuraminidase activity completely failed to activate F at pH 5.7 and was capable of only minimal triggering activity even at pH 8.0. These results demonstrate that neuraminidase activity impacts the extent of HPIV3-mediated fusion by releasing HN from contact with receptor. Any particular HN′s competence to promote F-mediated fusion depends on the balance between its inherent F-triggering efficacy and its receptor-attachment regulatory functions (binding and receptor cleavage).
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Paterson, Reay G., Michael L. Johnson, and Robert A. Lamb. "Paramyxovirus Fusion (F) Protein and Hemagglutinin–Neuraminidase (HN) Protein Interactions: Intracellular Retention of F and HN Does Not Affect Transport of the Homotypic HN or F Protein." Virology 237, no. 1 (1997): 1–9. http://dx.doi.org/10.1006/viro.1997.8759.
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