Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
The ORF4a protein of human coronavirus 229E functions as a viroporin that regulates viral production.
Zhang R
,
Wang K
,
Lv W
,
Yu W
,
Xie S
,
Xu K
,
Schwarz W
,
Xiong S
,
Sun B
.
???displayArticle.abstract???
In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li(+) partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.
Fig. 1. Sequence alignment and structure prediction of HCoV-229E ORF4a. (A) Amino acid sequence alignment of HCoV-229E ORF4a with SARS-CoV 3a using ClustalW2. The “*†indicates identical residues, the “:†indicates conserved substitution and the “.†indicates semi-conserved substitution. (B) Prediction of the transmembrane (TM) domains of the ORF4a protein. Three different programs were used to predict the TMs of ORF4a, including DAS, Phobius and TMHMM. The red letters indicate the putative TMs.
Fig. 2. The expression and subcellular localization of the HCoV-229E ORF4a protein. (A) The ORF4a protein was detected in HCoV-299E-infected Huh-7 cells using Western blot analysis. Huh-7 cells were infected with HCoV-229E at an MOI of 0.1 or mock-infected as a control. (B) Subcellular localization of the ORF4a protein in transfected Huh-7 cells. The ORF4a protein was detected with rabbit anti-HA antibody and visualized with Cy3-conjugated goat anti-rabbit antibody (red). ERGIC was detected with mouse anti-ERGIC-53 antibody (B-9) and visualized with Alexa Fluor 488-conjugated goat anti-mouse antibody (green). The nuclei were counterstained with DAPI (blue). Yellow signals in merged pictures show colocalization. White box and arrow correspond to colocalization analysis of fluorescence intensities (arbitrary units) of the dyes that were measured by ImageJ software, and shown next to the image. Bars represent 25 μm.
Fig. 3. The ORF4a protein forms homo-oligomers. (A) HEK293T cells were transfected with pCAGGS-ORF4a-HA or pCAGGS-ORF4a-Flag plasmids and subjected to immunoprecipitation with a monoclonal anti-Flag antibody. The associated HA-tagged ORF4a protein was detected using Western blot analysis with a polyclonal anti-HA antibody. (B) The HEK293 cells were transfected with pCAGGS-ORF4a-Flag. Cell lysates were immunoprecipitated with anti-Flag antibody and treated or not treated with β-mercaptoethanol (β-ME). The ORF4a proteins were detected by immunoblot analysis using the anti-Flag antibody. The arrows indicate bands corresponding to the monomer and putative oligomers.
Fig. 4. The ORF4a protein forms ion channels in Xenopus oocytes and yeast. (A) Representative current traces were recorded by two-electrode voltage clamp (TEVC) step from − 150 to + 30 mV in non-injected control oocytes and ORF4a-HA-expressing oocytes of the same batch. (B) The I/V relationship of voltage dependencies of steady-state currents in control oocytes (filled circles) and ORF4a-HA-expressing oocytes (filled squares). Current values were averaged across all oocyte batches tested. Data represent the mean ± SEM. (C) Complementation of a potassium uptake-deficient strain of S. cerevisiae with a pYES2-ORF4a-HA or pYES2 empty vector. The transformed yeast was grown on media containing 100 mM KCl or 0.2 mM KCl. Yeast was diluted as indicated and inoculated on the plates.
Fig. 5. Selectivity of ORF4a channel to different monovalent cations. (A) The I/V relationship of the currents conducted by ORF4a channel in the presence of different cations. The endogenous currents under identical conditions were subtracted. (B) Inward currents at â 150 mV were recorded by TEVC with different cations in the bath solution. (C) Conductance ratio (GX/GK, where X represents the other cations) is the slope conductance calculated from â 100 to â 150 mV. Data represent the mean ± SEM (n = 6â7).
Fig. 6. The suppression of ORF4a expression inhibits virus production. (A) siRNA targeting the HCoV-229E ORF4a gene (siORF4a) or control siRNA (siControl) was transfected into Huh-7 cells before virus infection. The suppression efficiency of siORF4a was detected using a Western blot assay 48 h post-infection. (B) Supernatants containing infectious virus particles from siRNA-treated cells were titered by TCID50 assay. Data represent the mean ± SD and were generated from three independent experiments (*P < 0.05, compared with siControl).
Adams,
Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2012).
2012, Pubmed
Adams,
Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2012).
2012,
Pubmed
Cai,
Putative caveolin-binding sites in SARS-CoV proteins.
2003,
Pubmed
Chan,
The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function.
2009,
Pubmed
Douglas,
Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism.
2009,
Pubmed
Eisenman,
Ionic selectivity revisited: the role of kinetic and equilibrium processes in ion permeation through channels.
1983,
Pubmed
Ewart,
The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels.
1996,
Pubmed
Fischer,
Viral channel-forming proteins.
2009,
Pubmed
Fischer,
Viral channel forming proteins - modeling the target.
2011,
Pubmed
Fischer,
Viral ion channels: structure and function.
2002,
Pubmed
Gazzarrini,
Chlorella virus ATCV-1 encodes a functional potassium channel of 82 amino acids.
2009,
Pubmed
,
Xenbase
Gladue,
Classical swine fever virus p7 protein is a viroporin involved in virulence in swine.
2012,
Pubmed
Gonzalez,
Viroporins.
2003,
Pubmed
Grice,
Ion channels formed by HIV-1 Vpu: a modelling and simulation study.
1997,
Pubmed
Griffin,
The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine.
2003,
Pubmed
Hamre,
A new virus isolated from the human respiratory tract.
1966,
Pubmed
Helenius,
Unpacking the incoming influenza virus.
1992,
Pubmed
Hsu,
Membrane potential depolarization as a triggering mechanism for Vpu-mediated HIV-1 release.
2010,
Pubmed
Hsu,
Mutual functional destruction of HIV-1 Vpu and host TASK-1 channel.
2004,
Pubmed
Hulit,
The cyclin D1 gene is transcriptionally repressed by caveolin-1.
2000,
Pubmed
Hyser,
Rotavirus disrupts calcium homeostasis by NSP4 viroporin activity.
2010,
Pubmed
Khoury,
Antiviral efficacy of the novel compound BIT225 against HIV-1 release from human macrophages.
2010,
Pubmed
Ksiazek,
A novel coronavirus associated with severe acute respiratory syndrome.
2003,
Pubmed
Lu,
Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release.
2006,
Pubmed
,
Xenbase
Luscombe,
A novel Hepatitis C virus p7 ion channel inhibitor, BIT225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-alpha-2b and nucleoside analogues.
2010,
Pubmed
Mangeat,
HIV-1 Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its beta-TrCP2-dependent degradation.
2009,
Pubmed
Masters,
The molecular biology of coronaviruses.
2006,
Pubmed
McIntosh,
Growth in suckling-mouse brain of "IBV-like" viruses from patients with upper respiratory tract disease.
1967,
Pubmed
Mehnert,
Biophysical characterization of Vpu from HIV-1 suggests a channel-pore dualism.
2008,
Pubmed
Mitchell,
Vpu antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.
2009,
Pubmed
Müller,
Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.
2010,
Pubmed
Nakamura,
Determination of key structural requirements of a K+ channel pore.
1997,
Pubmed
,
Xenbase
Neil,
An interferon-alpha-induced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein.
2007,
Pubmed
Nieva,
Viroporins: structure and biological functions.
2012,
Pubmed
Padhan,
Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin.
2007,
Pubmed
Park,
Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses.
2008,
Pubmed
Pavlović,
The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives.
2003,
Pubmed
Peiris,
Coronavirus as a possible cause of severe acute respiratory syndrome.
2003,
Pubmed
Pelkmans,
Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER.
2001,
Pubmed
Pinto,
Influenza virus M2 protein has ion channel activity.
1992,
Pubmed
,
Xenbase
Plugge,
A potassium channel protein encoded by chlorella virus PBCV-1.
2000,
Pubmed
,
Xenbase
Premkumar,
Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.
2004,
Pubmed
Raghava,
The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release.
2011,
Pubmed
Rota,
Characterization of a novel coronavirus associated with severe acute respiratory syndrome.
2003,
Pubmed
Roux,
Ion selectivity in channels and transporters.
2011,
Pubmed
Schwarz,
Emodin inhibits current through SARS-associated coronavirus 3a protein.
2011,
Pubmed
Song,
Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3.
2003,
Pubmed
Steinmann,
Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.
2007,
Pubmed
Sugrue,
Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel.
1991,
Pubmed
Suzuki,
The human polyoma JC virus agnoprotein acts as a viroporin.
2010,
Pubmed
Tan,
A novel severe acute respiratory syndrome coronavirus protein, U274, is transported to the cell surface and undergoes endocytosis.
2004,
Pubmed
van Boheemen,
Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans.
2012,
Pubmed
Van Damme,
The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein.
2008,
Pubmed
van der Hoek,
Identification of a new human coronavirus.
2004,
Pubmed
Wang,
PEDV ORF3 encodes an ion channel protein and regulates virus production.
2012,
Pubmed
,
Xenbase
Wang,
Viral proteins function as ion channels.
2011,
Pubmed
Woo,
Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia.
2005,
Pubmed
Wozniak,
Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
2010,
Pubmed
Xie,
DIDS blocks a chloride-dependent current that is mediated by the 2B protein of enterovirus 71.
2011,
Pubmed
,
Xenbase
Yount,
Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice.
2005,
Pubmed
Yu,
Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus.
2004,
Pubmed
Yuan,
Subcellular localization and membrane association of SARS-CoV 3a protein.
2005,
Pubmed
Zaki,
Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.
2012,
Pubmed
Zeng,
Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients.
2004,
Pubmed
