Article

Coronavirus E protein forms ion channels with functionally and structurally-involved membrane lipids.

Department of Physics, Laboratory of Molecular Biophysics, Universitat Jaume I, 12071 Castellón, Spain.
Virology (Impact Factor: 3.28). 07/2012; 432(2):485-94. DOI: 10.1016/j.virol.2012.07.005
Source: PubMed

ABSTRACT Coronavirus (CoV) envelope (E) protein ion channel activity was determined in channels formed in planar lipid bilayers by peptides representing either the transmembrane domain of severe acute respiratory syndrome CoV (SARS-CoV) E protein, or the full-length E protein. Both of them formed a voltage independent ion conductive pore with symmetric ion transport properties. Mutations N15A and V25F located in the transmembrane domain prevented the ion conductivity. E protein derived channels showed no cation preference in non-charged lipid membranes, whereas they behaved as pores with mild cation selectivity in negatively-charged lipid membranes. The ion conductance was also controlled by the lipid composition of the membrane. Lipid charge also regulated the selectivity of a HCoV-229E E protein derived peptide. These results suggested that the lipids are functionally involved in E protein ion channel activity, forming a protein-lipid pore, a novel concept for CoV E protein ion channel entity.

0 Bookmarks
 · 
98 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.
    PLoS Pathogens 08/2014; 10(8):e1004320. DOI:10.1371/journal.ppat.1004320 · 8.14 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The unfolded protein response (UPR) is a signal transduction cascade triggered by perturbation of the homeostasis of the endoplasmic reticulum (ER). UPR resolves ER stress by activating a cascade of cellular response including the induction of molecular chaperones, translational attenuation, ER-associated degradation and other mechanisms. Under prolonged and irremediable ER stress, however, UPR can also trigger apoptosis. Here we report that in cells infected with the avian coronavirus infectious bronchitis virus (IBV), ER stress was induced and the IRE1α-XBP1 pathway of UPR was activated. Knockdown and over-expression experiments demonstrated that IRE1α protects the infected cells from IBV-induced apoptosis, which required both its kinase and RNase activity. Our data also suggest that splicing of XBP1 mRNA by IRE1α appears to convert XBP1 from a pro-apoptotic XBP1u protein to a pro-survival XBP1s protein. Moreover, IRE1α antagonized IBV-induced apoptosis by modulating the phosphorylation status of the pro-apoptotic c-Jun N-terminal kinase (JNK) and the pro-survival RAC-alpha serine/threonine-protein kinase (Akt). Taken together, the ER stress sensor IRE1α is activated in IBV-infected cells and serves as a survival factor during coronavirus infection.
    Journal of Virology 08/2014; DOI:10.1128/JVI.02138-14 · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The small hydrophobic (SH) protein is a 64-amino acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the trasnmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity, although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. These latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin.
    Journal of Virology 10/2014; 88(20):11899-11914. DOI:10.1128/jvi.00839-14 · 4.65 Impact Factor

Full-text

Download
185 Downloads
Available from
Jun 2, 2014