Newcastle Disease Virus Entry into Chicken Macrophages via a pH-Dependent, Dynamin and Caveola-Mediated Endocytic Pathway That Requires Rab5

The cellular entry pathways and the mechanisms of Newcastle disease virus (NDV) entry into cells are poorly characterized. In this study, we demonstrated that chicken interferon-induced transmembrane protein 1 (chIFITM1) which is located in the early endosomes could limit the replication of NDV in chicken macrophage cell line HD11, suggesting the endocytic entry of NDV into chicken macrophages. Then, we presented a systematic study about the entry mechanism of NDV into chicken macrophages. First, we demonstrated that a low-pH condition and dynamin were required during NDV entry. However, NDV entry into chicken macrophages was independent of clathrin-mediated endocytosis. We also found that NDV entry was dependent on membrane cholesterol. The NDV entry and replication were significantly reduced by nystatin and Phorbol 12-myristate 13-acetate treatment, overexpression of dominant negative (DN) caveolin-1 or knockdown of caveolin-1, suggesting that NDV entry depends on caveola-mediated endocytosis. However, macropinocytosis did not play a role in NDV entry into chicken macrophages. Additionally, we found that Rab5, rather than Rab7, was involved in the entry and traffic of NDV. The colocalization of NDV with Rab5 and early endosome suggested that NDV virion was transported to early endosomes in a Rab5-dependent manner after internalization. Of particular note, the caveola-mediated endocytosis was also utilized by NDV to enter primary chicken macrophages. And NDV entered different cell types using different pathways. Collectively, our findings demonstrate for the first time that NDV virion enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway and Rab5 is involved in the traffic and location of NDV. IMPORTANCE Although the pathogenesis of Newcastle disease virus (NDV) has been extensively studied, the detailed mechanism of NDV entry into host cells is largely unknown. Macrophages are the first-line defenders of host defense against infection of pathogens. Chicken macrophages are considered as one of the main types of target cells during NDV infection. Here, we comprehensively investigated the entry mechanism of NDV in chicken macrophages. This is the first report to demonstrate that NDV enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway that requires Rab5. The result is important for our understanding of the entry of NDV in chicken macrophages, which will further advance the knowledge of NDV pathogenesis and provide useful clues for the development of novel preventive or therapeutic strategies against NDV infection. In addition, this information will contribute to our further understanding of pathogenesis with regard to other numbers of Avulavirus genus in Paramyxoviridae family.

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Newcastle disease virus may enter cells by caveolae-mediated endocytosis

Journal of General Virology ◽

10.1099/vir.0.82150-0 ◽

2007 ◽

Vol 88 (2) ◽

pp. 559-569 ◽

Cited By ~ 58

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.

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Incorporation of Functional HN-F Glycoprotein-Containing Complexes into Newcastle Disease Virus Is Dependent on Cholesterol and Membrane Lipid Raft Integrity

Journal of Virology ◽

10.1128/jvi.01119-07 ◽

2007 ◽

Vol 81 (19) ◽

pp. 10636-10648 ◽

Cited By ~ 16

Author(s):

Jason P. Laliberte ◽

Lori W. McGinnes ◽

Trudy G. Morrison

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Cell Attachment ◽

Particle Density ◽

Protein Composition ◽

Membrane Lipid ◽

Significant Loss ◽

Membrane Rafts ◽

Target Cells

ABSTRACT Newcastle disease virus assembles in plasma membrane domains with properties of membrane lipid rafts, and disruption of these domains by cholesterol extraction with methyl-β-cyclodextrin resulted in the release of virions with irregular protein composition, abnormal particle density, and reduced infectivity (J. P. Laliberte, L. W. McGinnes, M. E. Peeples, and T. G. Morrison, J. Virol. 80:10652-10662, 2006). In the present study, these results were confirmed using Niemann-Pick syndrome type C cells, which are deficient in normal membrane rafts due to mutations affecting cholesterol transport. Furthermore, cholesterol extraction of infected cells resulted in the release of virions that attached to target cells at normal levels but were defective in virus-cell membrane fusion. The reduced fusion capacity of particles released from cholesterol-extracted cells correlated with significant loss of HN-F glycoprotein-containing complexes detected in the virion envelopes of these particles and with detection of cell-associated HN-F protein-containing complexes in extracts of cholesterol-extracted cells. Extraction of cholesterol from purified virions had no effect on virus-cell attachment, virus-cell fusion, particle infectivity, or the levels of glycoprotein-containing complexes. Taken together, these results suggest that cholesterol and membrane rafts are required for the formation or maintenance of HN-F glycoprotein-containing complexes in cells but not the stability of preformed glycoprotein complexes once assembled into virions.

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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 ◽

10.1128/jvi.79.2.1180-1190.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 1180-1190 ◽

Cited By ~ 22

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.

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Role of Thiol/Disulfide Exchange in Newcastle Disease Virus Entry

Journal of Virology ◽

10.1128/jvi.01407-08 ◽

2008 ◽

Vol 83 (1) ◽

pp. 241-249 ◽

Cited By ~ 24

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.

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Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

Journal of Virology ◽

10.1128/jvi.00211-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 8108-8121 ◽

Cited By ~ 103

Author(s):

Mostafa Jarahian ◽

Carsten Watzl ◽

Philippe Fournier ◽

Annette Arnold ◽

Dominik Djandji ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Nk Cells ◽

Tumor Cells ◽

Disease Virus ◽

Newcastle Disease ◽

Nk Cell ◽

Target Cells ◽

Human Carcinoma ◽

Infected Cells ◽

Infected Tumor

ABSTRACT The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.

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α2-3- and α2-6- N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target cells

Glycoconjugate Journal ◽

10.1007/s10719-012-9431-0 ◽

2012 ◽

Vol 29 (7) ◽

pp. 539-549 ◽

Cited By ~ 21

Author(s):

Lorena Sánchez-Felipe ◽

Enrique Villar ◽

Isabel Muñoz-Barroso

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Sialic Acids ◽

Target Cells

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

Journal of Virology ◽

10.1128/jvi.80.6.2894-2903.2006 ◽

2006 ◽

Vol 80 (6) ◽

pp. 2894-2903 ◽

Cited By ~ 32

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.

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