scholarly journals Thiol/Disulfide Exchange Is Required for Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein

2006 ◽  
Vol 81 (5) ◽  
pp. 2328-2339 ◽  
Author(s):  
Surbhi Jain ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Cell Surface ◽  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Fusion Proteins ◽  
Protein Disulfide Isomerase ◽  
Viral Fusion ◽  
Disulfide Isomerase ◽  
F Protein ◽  
Viral Fusion Proteins ◽  
Protein Disulfide

ABSTRACT Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity—5′5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody—inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes.

Cell Surface Protein Disulfide-Isomerase Is Involved in the Shedding of Human Thyrotropin Receptor Ectodomain†

Biochemistry ◽  
1996 ◽  
Vol 35 (47) ◽  
pp. 14800-14805 ◽  
Author(s):  
Jacques Couët ◽  
Simon de Bernard ◽  
Hugues Loosfelt ◽  
Bertrand Saunier ◽  
Edwin Milgrom ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Disulfide Isomerase ◽  
Surface Protein ◽  
Cell Surface Protein ◽  
Thyrotropin Receptor ◽  
Disulfide Isomerase ◽  
Protein Disulfide

scholarly journals Conserved Leucines in N-Terminal Heptad Repeat HR1 of Envelope Fusion Protein F of Group II Nucleopolyhedroviruses Are Important for Correct Processing and Essential for Fusogenicity

2007 ◽  
Vol 82 (5) ◽  
pp. 2437-2447 ◽  
Author(s):  
Gang Long ◽  
Xiaoyu Pan ◽  
Just M. Vlak
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Leucine Zipper ◽  
Fusion Peptide ◽  
Class I ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
F Protein ◽  
Viral Fusion Proteins ◽  
Budded Virus

ABSTRACT The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F1. Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.

ADAMs and protein disulfide isomerase: the key to regulated cell-surface protein ectodomain shedding?

2010 ◽  
Vol 428 (3) ◽  
pp. e3-e5 ◽  
Author(s):  
Rosemary Bass ◽  
Dylan R. Edwards
Keyword(s):  
Cell Surface ◽  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Cell Signalling ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Proteolytic Processing ◽  
Surface Proteins ◽  
Disulfide Isomerase ◽  
Protein Disulfide

The ADAM disintegrin metalloproteinases (where ADAM is ‘a disintegrin and metalloproteinase’) are a family of transmembrane cell-surface proteins with essential roles in adhesion and proteolytic processing in all animals. The archetypal family member is ADAM17 {also known as TACE [TNFα (tumour necrosis factor α)-converting enzyme]}, which is involved in processing pro-TNFα and in the activation of ligands for the EGFR [EGF (epidermal growth factor) receptor], as well as cleavage of diverse cell-surface receptors and adhesion molecules. ADAM-mediated shedding is itself influenced via cell signalling pathways. In this issue of the Biochemical Journal, Willems et al. make the observation that phorbol ester activates shedding by ADAM17 by affecting the activity of PDI (protein disulfide isomerase). They propose that PDI maintains ADAM17 in an inactive ‘closed’ state and PMA stimulation generates ROS (reactive oxygen species) and thus an altered redox environment, which in turn inactivates PDI and allows ADAM17 to adopt an ‘open’ active conformation. This activation is accompanied by changes in disulfide bonds in the ADAM17 ectodomain. This is a novel and exciting finding that could help to unlock the actions of ADAM sheddases, as well as a host of other mechanisms that rely upon rapid alterations in protein conformation on the cell surface.

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.

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