scholarly journals Role of Thiol/Disulfide Exchange in Newcastle Disease Virus Entry

2008 ◽  
Vol 83 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Surbhi Jain ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Conformational Changes ◽  
Newcastle Disease ◽  
Disulfide Bonds ◽  
Early Stage ◽  
Protein S ◽  
Host Cells ◽  
Target Cells ◽  
F Protein

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.

scholarly journals 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

2005 ◽  
Vol 79 (2) ◽  
pp. 1180-1190 ◽  
Author(s):  
Jianrong Li ◽  
Vanessa R. Melanson ◽  
Anne M. Mirza ◽  
Ronald M. Iorio
Keyword(s):  
Newcastle Disease Virus ◽  
Conformational Change ◽  
Disease Virus ◽  
Cell Lines ◽  
Newcastle Disease ◽  
Energy Requirement ◽  
Target Cells ◽  
F Protein ◽  
Viral Hemagglutinin ◽  
Receptor Recognition

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.

scholarly journals Characterization of an Alternate Form of Newcastle Disease Virus Fusion Protein

2005 ◽  
Vol 79 (18) ◽  
pp. 11660-11670 ◽  
Author(s):  
Homer Pantua ◽  
Lori W. McGinnes ◽  
John Leszyk ◽  
Trudy G. Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Cell Fusion ◽  
Newcastle Disease ◽  
Host Cells ◽  
Classical Type ◽  
F Protein ◽  
Amino Terminal ◽  
Alternate Form

ABSTRACT The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes and infected COS-7 cells (J. Virol. 77:1951-1963, 2003). One form is the classical type 1 glycoprotein, while the other is a polytopic form in which approximately 200 amino acids of the amino-terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein-expressing cells, and antibodies specific for these sequences inhibited red blood cell fusion to hemagglutinin-neuraminidase and F protein-expressing cells, suggesting a role for surface-expressed CT sequences in cell-cell fusion. Extending these findings, we have found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of the F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single-cycle infections, as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed.

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 Inhibition of Receptor Binding Stabilizes Newcastle Disease Virus HN and F Protein-Containing Complexes

2006 ◽  
Vol 80 (6) ◽  
pp. 2894-2903 ◽  
Author(s):  
L. W. McGinnes ◽  
T. G. Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Receptor Binding ◽  
Newcastle Disease ◽  
Protein Complexes ◽  
Target Cells ◽  
Protein Cleavage ◽  
Protein Activity ◽  
Neuraminidase Treatment ◽  
F Protein

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.

scholarly journals Newcastle disease virus may enter cells by caveolae-mediated endocytosis

2007 ◽  
Vol 88 (2) ◽  
pp. 559-569 ◽  
Author(s):  
Celia Cantín ◽  
Javier Holguera ◽  
Laura Ferreira ◽  
Enrique Villar ◽  
Isabel Muñoz-Barroso
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Inhibitory Effect ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Virus Concentration ◽  
Double Labelling ◽  
Virus Adsorption ◽  
Labelling Immunofluorescence

The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses, is believed to occur by direct fusion at the plasma membrane through a pH-independent mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when drugs were added after virus adsorption. Cells treated with methyl β-cyclodextrin, a drug that sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus–cell binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies against the early endosome marker EEA1 revealed the localization of the virus in these intracellular structures. Using fluorescence microscopy, it was found that cell–cell fusion was enhanced at low pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway, suggesting that this pathway could be an alternative route for virus entry into cells.

scholarly journals Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Udaya S. Rangaswamy ◽  
Weijia Wang ◽  
Xing Cheng ◽  
Patrick McTamney ◽  
Danielle Carroll ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Receptor Binding ◽  
Newcastle Disease ◽  
Persistent Infection ◽  
Cleavage Site ◽  
Protein Cleavage ◽  
F Protein ◽  
Sialic Acid Binding

ABSTRACT Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses. IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of the HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity, and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also shed light on the critical balance between the F and HN proteins required for efficient NDV infection and their role in avian pathogenicity.

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