scholarly journals Mutations in the Fusion Peptide and Adjacent Heptad Repeat Inhibit Folding or Activity of the Newcastle Disease Virus Fusion Protein

2001 ◽  
Vol 75 (17) ◽  
pp. 7934-7943 ◽  
Author(s):  
Theresa A. Sergel ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Coiled Coil ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Proteolytic Cleavage ◽  
Heptad Repeat ◽  
Fusion Activity

ABSTRACT Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172–14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease virus fusion protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the “a” and “d” positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant proteins expressed on the cell surface were characterized for fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the “a” position interfered with proteolytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven “d” position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these “d” mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most “d” position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal fusion activity. That most of the “d” position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the prefusion form of the protein. We also found that mutations within the fusion peptide, at residue 128, can interfere with folding of the protein, implicating this region in folding of the molecule. No characterized mutation enhanced fusion.

scholarly journals Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus

2002 ◽  
Vol 83 (3) ◽  
pp. 623-629 ◽  
Author(s):  
Ming Yu ◽  
Enxiu Wang ◽  
Youfang Liu ◽  
Dianjun Cao ◽  
Ningyi Jin ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Large Scale ◽  
Heptad Repeat ◽  
Scale Production ◽  
F Protein ◽  
Helix Bundle ◽  
Fusion Inhibitors

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus–cell fusion inhibition activity (IC50 of 1·07–2·93 μM). The solubility of the GST–HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.

Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

2000 ◽  
Vol 20 (6) ◽  
pp. 597-612 ◽  
Author(s):  
Rebecca Ellis Dutch ◽  
Theodore S. Jardetzky ◽  
Robert A. Lamb
Keyword(s):  
Fusion Proteins ◽  
Ebola Virus ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Proteolytic Cleavage ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Hydrophobic Region ◽  
Viral Fusion Proteins

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.

scholarly journals Mutational Analysis of Heptad Repeats in the Membrane-Proximal Region of Newcastle Disease Virus HN Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 3630-3637 ◽  
Author(s):  
Judith Stone-Hulslander ◽  
Trudy G. Morrison
Keyword(s):  
Amino Acids ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Specific Interaction ◽  
Heptad Repeat ◽  
Sequence Motif ◽  
F Protein ◽  
Promotion Activity ◽  
Heptad Repeats

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.

scholarly journals Interacting Domains of the HN and F Proteins of Newcastle Disease Virus

2003 ◽  
Vol 77 (20) ◽  
pp. 11040-11049 ◽  
Author(s):  
Kathryn A. Gravel ◽  
Trudy G. Morrison
Keyword(s):  
Amino Acids ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Fusion Proteins ◽  
Specific Interaction ◽  
Point Mutations ◽  
Protein Sequences ◽  
Heptad Repeat ◽  
F Protein

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.

scholarly journals Mutational Analysis of the Membrane Proximal Heptad Repeat of the Newcastle Disease Virus Fusion Protein

Virology ◽  
2001 ◽  
Vol 289 (2) ◽  
pp. 343-352 ◽  
Author(s):  
Lori W. McGinnes ◽  
Theresa Sergel ◽  
Hong Chen ◽  
Ludwig Hamo ◽  
Steve Schwertz ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Mutational Analysis ◽  
Heptad Repeat ◽  
Virus Fusion

Acidic amino acids increase fusion activity in the specific fusion domain of Newcastle disease virus fusion protein

2013 ◽  
Vol 59 (9) ◽  
pp. 641-644 ◽  
Author(s):  
Guijie Ren ◽  
Yunlong Zhuang ◽  
Keli Tian ◽  
Huiyu Li ◽  
Xueqin Diao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Cell Fusion ◽  
Newcastle Disease ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Fusion Activity ◽  
Wild Type ◽  
F Protein ◽  
Fusion Domain

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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