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Title: Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

Abstract

Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six {alpha}-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host.

Authors:
 [1];  [2];  [3];  [2];  [3];  [4];  [4];  [5]
  1. Department of Preventive Veterinary, College of Veterinary Medicine, Northeast Agriculture University, 150030 Harbin (China)
  2. Laboratory of Molecular Immunology and Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080 (China)
  3. (China)
  4. China Agricultural University, Beijing 100094 (China)
  5. Department of Preventive Veterinary, College of Veterinary Medicine, Northeast Agriculture University, 150030 Harbin (China). E-mail: yijingli@163.com
Publication Date:
OSTI Identifier:
20713466
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 337; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2005.09.189; PII: S0006-291X(05)02226-6; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CHROMATOGRAPHY; DRUGS; GELS; IN VITRO; POLYPEPTIDES

Citation Formats

Ma Guangpeng, Feng Youjun, Graduate School of the Chinese Academy of Sciences, Gao Feng, China Agricultural University, Beijing 100094, Wang Jinzi, Liu Cheng, and Li Yijing. Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.09.189.
Ma Guangpeng, Feng Youjun, Graduate School of the Chinese Academy of Sciences, Gao Feng, China Agricultural University, Beijing 100094, Wang Jinzi, Liu Cheng, & Li Yijing. Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core. United States. doi:10.1016/j.bbrc.2005.09.189.
Ma Guangpeng, Feng Youjun, Graduate School of the Chinese Academy of Sciences, Gao Feng, China Agricultural University, Beijing 100094, Wang Jinzi, Liu Cheng, and Li Yijing. Fri . "Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core". United States. doi:10.1016/j.bbrc.2005.09.189.
@article{osti_20713466,
title = {Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core},
author = {Ma Guangpeng and Feng Youjun and Graduate School of the Chinese Academy of Sciences and Gao Feng and China Agricultural University, Beijing 100094 and Wang Jinzi and Liu Cheng and Li Yijing},
abstractNote = {Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six {alpha}-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host.},
doi = {10.1016/j.bbrc.2005.09.189},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 337,
place = {United States},
year = {Fri Dec 02 00:00:00 EST 2005},
month = {Fri Dec 02 00:00:00 EST 2005}
}
  • A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-{delta}E) has been engineered. This deletion mutant only grows in cells expressing E protein (E{sup +} cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-{delta}E infected BHK-pAPN-E{sup -} cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E{sup -} cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-{delta}E virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless,more » full virion maturation was blocked. Studies of the rTGEV-{delta}E subcellular localization by confocal and immunoelectron microscopy in infected E{sup -} cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation.« less
  • Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimermore » of coiled-coil heterodimer (six {alpha}-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection.« less
  • Highlights: •TGEV infection induced ROS accumulation. •ROS accumulation is involved in TGEV-induced mitochondrial integrity impairment. •ROS is associated with p53 activation and apoptosis occurrence in TGEV-infected cells. -- Abstract: Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potentialmore » in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-L-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.« less
  • Two approaches were taken to examine the genome structure of the porcine transmissible gastroenteritis coronavirus (TGEV). The first approach was designed to compare the degree of nucleotide sequence homology between the genomes of TGEV and the bovine enteric coronavirus (BCV), a member of an antigenically distinct coronavirus subgroup. For this study, the RNA genomes of TGEV and BCV were separately metabolically labeled with /sup 32/P-orthophosphate, and analyzed by two-dimensional T1 oligonucleotide fingerprinting. From an analysis of comigrating oligonucleotides in a mixing experiment, it was determined that TGEV and BCV share a sequence homology of approximately 91%. This degree of homologymore » supports the notion that the two viruses, while antigenically dissimilar, arose from a common ancestor. It further raises the question of whether conserved sequences might be confined to restricted regions of the genome allowing identification of conserved functional domains. The second approach was designed to systematically examine the primary structure of the TGEV genome thereby allowing rigorous genetic analyses. For this approach, the 3' end of the 20 Kb TGEV genome was cloned and sequenced, and the properties of two genes were deduced and analyzed.« less
  • The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressedmore » in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii.« less