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 Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus

1998 ◽  
Vol 72 (5) ◽  
pp. 3789-3795 ◽  
Author(s):  
Zengji Li ◽  
Theresa Sergel ◽  
Enal Razvi ◽  
Trudy Morrison
Keyword(s):  
Amino Acids ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Syncytium Formation ◽  
Dominant Negative ◽  
Wild Type ◽  
F Protein ◽  
Type Protein ◽  
Wild Type Protein

ABSTRACT The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.

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.

scholarly journals Mutations in the Ectodomain of Newcastle Disease Virus Fusion Protein Confer a Hemagglutinin-Neuraminidase-Independent Phenotype

2009 ◽  
Vol 84 (2) ◽  
pp. 1066-1075 ◽  
Author(s):  
Juan Ayllón ◽  
Enrique Villar ◽  
Isabel Muñoz-Barroso
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Host Cells ◽  
Heptad Repeat ◽  
Wild Type ◽  
Dye Transfer ◽  
F Protein ◽  
C Terminus ◽  
The Stability

ABSTRACT The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.

scholarly journals Mutated Form of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Interacts with the Homologous Fusion Protein despite Deficiencies in both Receptor Recognition and Fusion Promotion

2004 ◽  
Vol 78 (10) ◽  
pp. 5299-5310 ◽  
Author(s):  
Jianrong Li ◽  
Edward Quinlan ◽  
Anne Mirza ◽  
Ronald M. Iorio
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Viral Entry ◽  
Newcastle Disease ◽  
Active Site ◽  
Specific Interaction ◽  
Cellular Receptors ◽  
Dimer Interface ◽  
F Protein ◽  
Receptor Recognition

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.

scholarly journals 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

2004 ◽  
Vol 78 (23) ◽  
pp. 13053-13061 ◽  
Author(s):  
Vanessa R. Melanson ◽  
Ronald M. Iorio
Keyword(s):  
Amino Acid ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Amino Acid Substitutions ◽  
Globular Domain ◽  
Viral Glycoprotein ◽  
Specific Domain ◽  
F Protein ◽  
Heptad Repeats

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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