scholarly journals Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence

2012 ◽  
Vol 93 (9) ◽  
pp. 1965-1975 ◽  
Author(s):  
Y. X. Setoh ◽  
N. A. Prow ◽  
J. Hobson-Peters ◽  
M. Lobigs ◽  
P. R. Young ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Viral Particle ◽  
Cell Surface Expression ◽  
Antigenic Structure ◽  
West Nile ◽  
E Protein

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNVNY99) and a weakly virulent Australian subtype (WNVKUN). Five amino acid differences occur in WNVNY99 compared with WNVKUN (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNVNY99 prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNVKUN prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNVKUN prM to the same level as that of WNVNY99, and enhanced the secretion of WNVKUN prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNVKUN infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

scholarly journals A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection

2008 ◽  
Vol 89 (12) ◽  
pp. 3063-3072 ◽  
Author(s):  
Jody Hobson-Peters ◽  
Philip Toye ◽  
Melissa D. Sánchez ◽  
Katharine N. Bossart ◽  
Lin-Fa Wang ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Fusion Protein ◽  
Synthetic Peptides ◽  
Mammalian Cells ◽  
Protein Glycosylation ◽  
The West ◽  
West Nile ◽  
High Sequence Identity ◽  
Recombinant Peptide ◽  
E Protein

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147–165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

Galectin-lattice sustains function of cationic amino acid transporter and insulin secretion of pancreatic β cells

2020 ◽  
Vol 167 (6) ◽  
pp. 587-596 ◽  
Author(s):  
Kento Maeda ◽  
Masayoshi Tasaki ◽  
Yukio Ando ◽  
Kazuaki Ohtsubo
Keyword(s):  
Insulin Secretion ◽  
Amino Acid ◽  
Cell Surface ◽  
Amino Acid Transporter ◽  
Cell Surface Expression ◽  
Cationic Amino Acid Transporter ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Cationic Amino Acid ◽  
Acid Transporter

Abstract Maintenance of cell surface residency and function of glycoproteins by lectins are essential for regulating cellular functions. Galectins are β-galactoside-binding lectins and form a galectin-lattice, which regulates stability, clustering, membrane sub-domain localization and endocytosis of plasmalemmal glycoproteins. We have previously reported that galectin-2 (Gal-2) forms a complex with cationic amino acid transporter 3 (CAT3) in pancreatic β cells, although the biological significance of the molecular interaction between Gal-2 and CAT3 has not been elucidated. In this study, we demonstrated that the structure of N-glycan of CAT3 was either tetra- or tri-antennary branch structure carrying β-galactosides, which works as galectin-ligands. Indeed, CAT3 bound to Gal-2 using β-galactoside epitope. Moreover, the disruption of the glycan-mediated bindings between galectins and CAT3 significantly reduced cell surface expression levels of CAT3. The reduced cell surface residency of CAT3 attenuated the cellular arginine uptake activities and subsequently reduced nitric oxide production, and thus impaired the arginine-stimulated insulin secretion of pancreatic β cells. These results indicate that galectin-lattice stabilizes CAT3 by preventing endocytosis to sustain the arginine-stimulated insulin secretion of pancreatic β cells. This provides a novel cell biological insight into the endocrinological mechanism of nutrition metabolism and homeostasis.

scholarly journals SARS-CoV-2 envelope protein topology in eukaryotic membranes

Open Biology ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 200209 ◽  
Author(s):  
Gerard Duart ◽  
Mª Jesús García-Murria ◽  
Brayan Grau ◽  
José M. Acosta-Cáceres ◽  
Luis Martínez-Gil ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Mammalian Cells ◽  
E Proteins ◽  
Virus Envelope ◽  
Protein Topology ◽  
Membrane Protein Topology ◽  
E Protein ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Cytoplasmic Side

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Nt lum /Ct cyt ). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.

scholarly journals IRE1α kinase–mediated unconventional protein secretion rescues misfolded CFTR and pendrin

2020 ◽  
Vol 6 (8) ◽  
pp. eaax9914
Author(s):  
Hak Park ◽  
Dong Hoon Shin ◽  
Ju-Ri Sim ◽  
Sowon Aum ◽  
Min Goo Lee
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Protein Misfolding ◽  
Mammalian Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Downstream Effector ◽  
Mouse Colon ◽  
Δf508 Cftr ◽  
Kinase Pathway

The most prevalent pathogenic mutations in the CFTR (ΔF508) and SLC26A4/pendrin (p.H723R), which cause cystic fibrosis and congenital hearing loss, respectively, evoke protein misfolding and subsequent defects in their cell surface trafficking. Here, we report that activation of the IRE1α kinase pathway can rescue the cell surface expression of ΔF508-CFTR and p.H723R-pendrin through a Golgi-independent unconventional protein secretion (UPS) route. In mammalian cells, inhibition of IRE1α kinase, but not inhibition of IRE1α endonuclease and the downstream effector XBP1, inhibited CFTR UPS. Treatment with the IRE1α kinase activator, (E)-2-(2-chlorostyryl)-3,5,6-trimethyl-pyrazine (CSTMP), rescued cell surface expression and functional activity of ΔF508-CFTR and p.H723R-pendrin. Treatment with a nontoxic dose of CSTMP to ΔF508-CFTR mice restored CFTR surface expression and CFTR-mediated anion transport in the mouse colon. These findings suggest that UPS activation via IRE1α kinase is a strategy to treat diseases caused by defective cell surface trafficking of membrane proteins, including ΔF508-CFTR and p.H723R-pendrin.

scholarly journals Cell surface recycling in yeast: mechanisms and machineries

2016 ◽  
Vol 44 (2) ◽  
pp. 474-478 ◽  
Author(s):  
Chris MacDonald ◽  
Robert C. Piper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Genetic System ◽  
Integral Membrane Protein ◽  
Protein Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

scholarly journals Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e45851 ◽  
Author(s):  
Madhuri Namekar ◽  
Mukesh Kumar ◽  
Maile O'Connell ◽  
Vivek R. Nerurkar
Keyword(s):  
West Nile Virus ◽  
Heat Inactivation ◽  
West Nile ◽  
E Protein ◽  
Microsphere Immunoassay

scholarly journals A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells

2017 ◽  
Vol 240 ◽  
pp. 14-20 ◽  
Author(s):  
Shintaro Kobayashi ◽  
Kentaro Yoshii ◽  
Minato Hirano ◽  
Memi Muto ◽  
Hiroaki Kariwa
Keyword(s):  
West Nile Virus ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
West Nile
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