Zhang R et al. (2014), The ORF4a protein of human coronavirus 229E fun...

XB-ART-48015

Biochim Biophys Acta 2014 Apr 01;18384:1088-95. doi: 10.1016/j.bbamem.2013.07.025.

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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 .

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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.

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Species referenced: Xenopus
Genes referenced: pycard

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	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 − 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 − 100 to − 150 mV. Data represent the mean ± SEM (n = 6–7).
	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).

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