Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein

Abstract

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones, polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein, respectively, were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence, immunoelectron microscopy and cell surface protein labeling. In addition, measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed.

Fig. 1

Characterization of mAbs specific for SARS-CoV E protein. (A) E protein sequence is divided into three domains: the amino terminal (N-terminal), the transmembrane and the carboxy terminal (C-terminal). Gray letters represent hydrophobic amino acids, and black letters indicate hydrophilic amino acids. Jamesson–Wolf antigenic index (DNA-STAR, Lasergene) is shown below the amino acid sequence. Positive values in the graph represent high antigenicity indexes whereas negative values represent low antigenicity indexes. The highest antigenic regions of the protein are shown in gray boxes. (B) Immunofluorescences of SARS-CoV-infected Vero E6 cells. Vero E6 cells were grown on coverslips and infected at an moi of 0.3 with rSARS-CoV wt (wt) or with rSARS-CoV-∆E (∆E) as a control. At 24 hpi cells were fixed with 4% paraformaldehyde, and labeled with E protein specific antibodies (green). Rabbit polyclonal antibody specific for E protein (pAb α E) was used as a control. mAbs are numbered from E1 to E5. (C) Western blot analysis of SARS-CoV-infected Vero E6 cells. Vero E6 cells were infected at an moi of 0.3 with rSARS-CoV wt (wt) or with rSARS-CoV-∆E (∆E) as a control. At 24 hpi cell lysates were collected and probed with mAbs specific for E protein (E1 to E5) or with a polyclonal antibody used as a control (pAb α E). mAbs isotype is indicated bellow Western blot results (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Epitope mapping of SARS-CoV E protein specific mAbs and pAbs by PEPSCAN. (A) 34 peptides of 10 amino acids each covering the full length SARS-CoV E protein were synthesized on a cellulose membrane. The peptides overlapped contiguous peptides by 8 amino acids. (A) E protein derived peptides were probed by Western blot using a rabbit polyclonal antibody specific for E protein (pAb α E) or using the specific mAbs (E1 to E5). As controls, primary antibodies were omitted and goat secondary antibody coupled to HRP specific for rabbit (G α R HRP) and rabbit secondary antibody coupled to HRP specific for mouse (R α M HRP) were used alone. (B) Epitope mapping of rabbit polyclonal antibody specific for E protein amino terminal domain (pAb α ENT). As a control, a rabbit specific secondary antibody coupled to HRP (G α R HRP) was probed alone.

Fig. 3

Subcellular localization of SARS-CoV E protein analysis by immunofluorescence. Vero E6 cells were grown on coverslips and transfected with a plasmid encoding E protein (pcDNA-E) or infected with rSARS-CoV wt. Cells were fixed with 4% paraformaldehyde at 8 and 24 h post transfection (hpt) or post infection (hpi). (A) Cells were labeled with E protein (green) or ERGIC53 ERGIC marker (red) specific antibodies. (B) Cells were labeled with E protein (green) or PDI ER marker (red) specific antibodies. (C) E protein was labeled in green and plasma membrane (cadherin) was labeled in red using specific antibodies. Nuclei were stained with DAPI in all cases (blue).

Fig. 3

Subcellular localization of SARS-CoV E protein analysis by immunofluorescence. Vero E6 cells were grown on coverslips and transfected with a plasmid encoding E protein (pcDNA-E) or infected with rSARS-CoV wt. Cells were fixed with 4% paraformaldehyde at 8 and 24 h post transfection (hpt) or post infection (hpi). (A) Cells were labeled with E protein (green) or ERGIC53 ERGIC marker (red) specific antibodies. (B) Cells were labeled with E protein (green) or PDI ER marker (red) specific antibodies. (C) E protein was labeled in green and plasma membrane (cadherin) was labeled in red using specific antibodies. Nuclei were stained with DAPI in all cases (blue).

Fig. 4

Analysis of SARS-CoV E protein subcellular localization by immunoelectron microscopy. Vero E6 cells were infected with rSARS-CoV wt or rSARS-CoV-∆E. At 16 hpi cells were fixed with 4% paraformaldehyde and 0.125% glutaraldehyde, scrapped and pelleted. Pellets were processed and sectioned. (A) Ultrathin cryosections were incubated with mAb E5 and an anti-mouse secondary antibody labeled with 10 nm colloidal gold. PM (plasma membrane), VF (Viral factories). Bars 200 nm. (B) Around 800 gold dots were counted in several images of rSARS-CoV-∆E or rSARS-CoV wt-infected cells. Gold labeling is represented as gold dots per surface units (10 μm²) at the plasma membrane (PM), the viral factories (VF) and at unspecific locations (UN) such as the cell nucleus or mitochondrion, which represent the background displayed by the antibody. Statistically significant data are indicated with two asterisks (Student's t-test p-value < 0.01).

Fig. 5

Analysis of SARS-CoV E protein presence at the plasma membrane by cell surface protein biotinylation and purification assay. Vero E6 cells were either mock transfected, transfected with a plasmid encoding E protein (pcDNA-E) or with an empty plasmid as a control (pcDNA), or infected with rSARS-CoV wt (wt) or with rSARS-CoV-∆E (∆E) as a control. Plasma membrane proteins were biotin labeled and cells were lysed. Cell lysates (CL) were incubated with an avidin resin, and biotinylated cell surface proteins were isolated (B). Non-biotinylated proteins were discarded and eluted in the flow through (FT). CL, FT and B fractions were analyzed by Western blot using antibodies specific for the intracellular proteins β-actin and GRP78, the plasma membrane protein cadherin and SARS-CoV E protein.

Fig. 6

Whole-cell patch clamp. HEK-293T cells were mock transfected (Mock), transfected with an empty plasmid (pcDNA) or transfected with a plasmid encoding SARS-CoV E protein (pcDNA-E). At 24 hpt cells were voltage clamped, and stepped from − 160 mV to + 140 mV in 20 mV increments with pulses of 40 ms from a resting potential of − 40 mV. Current intensity was plotted against voltage. Currents values were measured at 30 ms, standard deviations are shown. Lower panels show examples of original recordings from each cell type.

Fig. 7

Identification of proteins interacting with SARS-CoV E protein. Vero E6 cell extracts from mock-infected cells (Mock) or cells infected with rSARS-CoV wt or rSARS-CoV-EtagCt (E-tag) were used in a double affinity chromatography. Purified proteins were detected by using Coomassie blue gels. Bands were excised from gels and identified by mass spectrometry. Arrows point Na⁺/K⁺ ATPase alpha 1 subunit and stomatin protein bands.

Fig. 8

SARS-CoV E protein topology. Vero E6 cells expressing E protein alone (pcDNA-E) or in an infection context (rSARS-CoV wt) were alternatively processed with no permeabilization (NP), selectively permeabilizing plasma membrane by using digitonin or permeabilizing all cell membranes by using Triton X-100. Specific antibodies were used to label the E protein carboxy terminal domain (ECT, green), E protein amino terminal domain (ENT, green), the cytosolic protein β-tubulin (red) and the luminal ER protein PDI (red). Nuclei were stained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)