scholarly journals Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus

1990 ◽  
Vol 10 (2) ◽  
pp. 449-457
Author(s):  
C Wilson ◽  
R Gilmore ◽  
T Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cytoplasmic Domain ◽  
Mutant Protein ◽  
Membrane Insertion ◽  
Type Ii ◽  
Wild Type ◽  
Amino Terminal ◽  
Nascent Chain

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a type II glycoprotein oriented in the plasma membrane with its amino terminus in the cytoplasm and its carboxy terminus external to the cell. We have previously shown that the membrane insertion of HN protein requires signal recognition particle SRP, occurs cotranslationally, and utilizes the same GTP-dependent step that has been described for secretory proteins, type I proteins, and multispanning proteins (C. Wilson, R. Gilmore, and T. Morrison, Mol. Cell. Biol. 7:1386-1392, 1987; C. Wilson, T. Connolly, T. Morrison, and R. Gilmore, J. Cell Biol. 107:69-77, 1988). The role of the amino-terminal cytoplasmic domain in the faithful membrane insertion of this type II protein was explored by characterizing the membrane integration of a mutant lacking 23 of the 26 amino acids of the cytoplasmic domain. The mutant protein was able to interact with SRP, resulting in translation inhibition, membrane targeting, and membrane translocation, but the efficiency of translocation was considerably lower than for the wild-type HN protein. In addition, a significant proportion of the mutant protein synthesized in the presence of SRP and microsomal membranes was associated with the membrane in an EDTA- and alkali-insensitive manner yet integrated into membranes with its carboxy-terminal domain on the cytoplasmic side of membrane vesicles. Membrane-integrated molecules with this reverse orientation were not detected when the mutant protein was synthesized in the absence of SRP or a functional SRP receptor. Truncated mRNAs encoding amino-terminal segments of the wild-type and mutant proteins were translated to prepare ribosomes bearing arrested nascent chains. The arrested mutant nascent chain, in contrast to the wild-type nascent chain, was also able to insert into membranes in a GTP- and SRP-independent manner. Results suggest that the cytoplasmic domain plays a role in the proper membrane insertion of this type II glycoprotein.

scholarly journals Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

1990 ◽  
Vol 10 (2) ◽  
pp. 449-457 ◽  
Author(s):  
C Wilson ◽  
R Gilmore ◽  
T Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cytoplasmic Domain ◽  
Mutant Protein ◽  
Membrane Insertion ◽  
Type Ii ◽  
Wild Type ◽  
Amino Terminal ◽  
Nascent Chain

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a type II glycoprotein oriented in the plasma membrane with its amino terminus in the cytoplasm and its carboxy terminus external to the cell. We have previously shown that the membrane insertion of HN protein requires signal recognition particle SRP, occurs cotranslationally, and utilizes the same GTP-dependent step that has been described for secretory proteins, type I proteins, and multispanning proteins (C. Wilson, R. Gilmore, and T. Morrison, Mol. Cell. Biol. 7:1386-1392, 1987; C. Wilson, T. Connolly, T. Morrison, and R. Gilmore, J. Cell Biol. 107:69-77, 1988). The role of the amino-terminal cytoplasmic domain in the faithful membrane insertion of this type II protein was explored by characterizing the membrane integration of a mutant lacking 23 of the 26 amino acids of the cytoplasmic domain. The mutant protein was able to interact with SRP, resulting in translation inhibition, membrane targeting, and membrane translocation, but the efficiency of translocation was considerably lower than for the wild-type HN protein. In addition, a significant proportion of the mutant protein synthesized in the presence of SRP and microsomal membranes was associated with the membrane in an EDTA- and alkali-insensitive manner yet integrated into membranes with its carboxy-terminal domain on the cytoplasmic side of membrane vesicles. Membrane-integrated molecules with this reverse orientation were not detected when the mutant protein was synthesized in the absence of SRP or a functional SRP receptor. Truncated mRNAs encoding amino-terminal segments of the wild-type and mutant proteins were translated to prepare ribosomes bearing arrested nascent chains. The arrested mutant nascent chain, in contrast to the wild-type nascent chain, was also able to insert into membranes in a GTP- and SRP-independent manner. Results suggest that the cytoplasmic domain plays a role in the proper membrane insertion of this type II glycoprotein.

scholarly journals Evidence for Mixed Membrane Topology of the Newcastle Disease Virus Fusion Protein

2003 ◽  
Vol 77 (3) ◽  
pp. 1951-1963 ◽  
Author(s):  
Lori W. McGinnes ◽  
Julie N. Reitter ◽  
Kathy Gravel ◽  
Trudy G. Morrison
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Cytoplasmic Domain ◽  
Classical Type ◽  
F Protein ◽  
Amino Terminal ◽  
Protease Digestion ◽  
A Cell

ABSTRACT The synthesis of the Newcastle disease virus (NDV) fusion (F) protein in a cell-free protein-synthesizing system containing membranes was characterized. The membrane-associated products were in at least two different topological forms with respect to the membranes. The properties of one form were consistent with the expected membrane insertion as a classical type 1 glycoprotein. This form of the protein was fully glycosylated, and sequences amino terminal to the transmembrane domain were protected from protease digestion by the membranes. The second form of membrane-associated F protein was partially glycosylated and partially protected from protease digestion by the membranes. Protease digestion resulted in a 23-kDa protease-protected polypeptide derived from F2 sequences and sequences from the amino-terminal end of the F1 domain. Furthermore, a 10-kDa polypeptide derived from the cytoplasmic domain (CT) was also protected from protease digestion by the membranes. Protease resistance of the 23- and 10-kDa polypeptides suggested that this second form of F protein inserted in membranes in a polytopic conformation with both the amino-terminal end and the carboxyl-terminal end translocated across membranes. To determine if this second form of the fusion protein could be found in cells expressing the F protein, two different approaches were taken. A polypeptide with the size of the partially translocated F protein was detected by Western analysis of proteins in total-cell extracts of NDV strain B1 (avirulent)-infected Cos-7 cells. Using antibodies raised against a peptide with sequences from the cytoplasmic domain, CT sequences were detected on surfaces of F protein-expressing Cos-7 cells by immunofluorescence and by flow cytometry. This antibody also inhibited the fusion of red blood cells to cells expressing F and HN proteins. These results suggest that NDV F protein made both in a cell-free system and in Cos-7 cells may exist in two topological forms with respect to membranes and that the second form of the protein may be involved in cell-cell fusion.

scholarly journals Newcastle Disease Virus HN Protein Alters the Conformation of the F Protein at Cell Surfaces

2002 ◽  
Vol 76 (24) ◽  
pp. 12622-12633 ◽  
Author(s):  
Lori W. McGinnes ◽  
Kathryn Gravel ◽  
Trudy G. Morrison
Keyword(s):  
Protein Expression ◽  
Newcastle Disease Virus ◽  
Red Blood Cells ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Blood Cells ◽  
Wild Type ◽  
Cell Surfaces ◽  
F Protein ◽  
Amino Terminal

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.

scholarly journals Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.

1990 ◽  
Vol 111 (1) ◽  
pp. 31-44 ◽  
Author(s):  
M K Spriggs ◽  
P L Collins
Keyword(s):  
Signal Sequence ◽  
Active Form ◽  
Cytoplasmic Domain ◽  
Biologically Active ◽  
Membrane Insertion ◽  
Type I ◽  
Type Ii ◽  
Membrane Anchor ◽  
Amino Terminal ◽  
Signal Anchor

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

scholarly journals Safety and Clinical Usage of Newcastle Disease Virus in Cancer Therapy

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Han Yuen Lam ◽  
Swee Keong Yeap ◽  
Mehdi Rasoli ◽  
Abdul Rahman Omar ◽  
Khatijah Yusoff ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Human Tumor ◽  
Wild Type ◽  
Anticancer Effect ◽  
Human Tumor Cells ◽  
Immunological Effects ◽  
Oncolytic Effect

Newcastle disease virus (NDV) is an avian virus that causes deadly infection to over 250 species of birds, including domestic and wild-type, thus resulting in substantial losses to the poultry industry worldwide. Many reports have demonstrated the oncolytic effect of NDV towards human tumor cells. The interesting aspect of NDV is its ability to selectively replicate in cancer cells. Some of the studies have undergone human clinical trials, and favorable results were obtained. Therefore, NDV strains can be the potential therapeutic agent in cancer therapy. However, investigation on the therapeutic perspectives of NDV, especially human immunological effects, is still ongoing. This paper provides an overview of the current studies on the cytotoxic and anticancer effect of NDV via direct oncolysis effects or immune stimulation. Safety of NDV strains applied for cancer immunotherapy is also discussed in this paper.

scholarly journals Translation and membrane insertion of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

1987 ◽  
Vol 7 (4) ◽  
pp. 1386-1392 ◽  
Author(s):  
C Wilson ◽  
R Gilmore ◽  
T Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Signal Recognition Particle ◽  
Trypsin Digestion ◽  
Membrane Insertion ◽  
Signal Recognition ◽  
Free System ◽  
Infected Cells ◽  
Made In

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.

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 Newcastle Disease Virus (NDV)-Based Assay Demonstrates Interferon-Antagonist Activity for the NDV V Protein and the Nipah Virus V, W, and C Proteins

2003 ◽  
Vol 77 (2) ◽  
pp. 1501-1511 ◽  
Author(s):  
Man-Seong Park ◽  
Megan L. Shaw ◽  
Jorge Muñoz-Jordan ◽  
Jerome F. Cros ◽  
Takaaki Nakaya ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Nipah Virus ◽  
Terminal Region ◽  
Ns1 Protein ◽  
Antagonist Activity ◽  
V Protein ◽  
Amino Terminal ◽  
C Proteins

ABSTRACT We have generated a recombinant Newcastle disease virus (NDV) that expresses the green fluorescence protein (GFP) in infected chicken embryo fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment of cells with chicken alpha/beta IFN (IFN-α/β) completely blocks viral GFP expression. Prior transfection of plasmid DNA induces an IFN response in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors of the IFN-α/β system, including the influenza A virus NS1 protein and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore conclude that the NDV-GFP virus could be used to screen proteins expressed from plasmids for the ability to counteract the host cell IFN response. Using this system, we show that expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-GFP replication in the face of the transfection-induced IFN response. The V and W proteins of Nipah virus, a highly lethal pathogen in humans, also block activation of an IFN-inducible promoter in primate cells. Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical to the amino terminus of Nipah virus W, is sufficient to exert the IFN-antagonist activity. In contrast, the anti-IFN activity of the NDV V protein appears to be located in the carboxy-terminal region of the protein, a region implicated in the IFN-antagonist activity exhibited by the V proteins of mumps virus and human parainfluenza virus type 2.

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