SARS coronavirus E protein forms cation-selective ion channels

Abstract

Severe Acute Respiratory Syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). Coronaviruses including SARS-CoV encode an envelope (E) protein, a small, hydrophobic membrane protein. We report that, in planar lipid bilayers, synthetic peptides corresponding to the SARS-CoV E protein forms ion channels that are more permeable to monovalent cations than to monovalent anions. Affinity-purified polyclonal antibodies recognizing the N-terminal 19 residues of SARS-CoV E protein were used to establish the specificity of channel formation by inhibiting the ion currents generated in the presence of the E protein peptides.

Fig. 1

SARS-CoV full-length and N-terminal E protein mass spectral analysis. Full-length E protein shows a predominant peak at m/z ratio of 8360.1, the expected molecular weight. The N-terminal peptide shows a prevalent peak at 4422.3 m/z, peaks of decreasing intensity are present, corresponding to products sequentially truncated by single amino acid residues.

Fig. 2

SARS-CoV E protein ion channel currents in lipid bilayers. The CIS chamber contained 500 mM NaCl, 5 mM HEPES buffer (pH 7.2); the TRANS chamber contained 50 mM NaCl, 5 mM HEPES buffer (pH 7.2). (A) Sample traces of raw current data, filtered at 100 Hz. The closed state is shown as a broken line, openings are deviations from the line. Scale bars are 200 ms and 5 pA. Potential was held at −88 or −48 mV. Conductances were 52 or 47 pS, respectively. (B) All points histograms of currents shown in A at −88 and C at −48 mV.

Fig. 3

SARS-CoV E protein ion channel activity observed in NaCl or KCl solutions. Data filtered at 100 Hz. (A) The CIS chamber contained 500 mM NaCl, 5 mM HEPES buffer (pH 7.2); the TRANS chamber contained 50 mM NaCl, 5 mM HEPES buffer (pH 7.2). The CIS chamber was earthed and the TRANS chamber was held at various potentials between −88 and +112 mV. The closed state is shown as a broken line, openings are deviations from the line. Scale bars are 200 ms and 10 pA. (B) IV relationship in NaCl solution average current measured against holding potential (mV). (C) The CIS chamber contained 500 mM KCl, 5 mM HEPES buffer (pH 7.2); the TRANS chamber contained 50 mM KCl in 5 mM HEPES buffer (pH 7.2). The CIS chamber was earthed and the TRANS chamber was held at various potentials between −89 and +91 mV. The closed state is shown as a broken line, openings are deviations from the line. Scale bars are 200 ms and 10 pA. (D) IV relationship in KCl solution, average current measured against holding potential (mV).

Fig. 4

SARS-CoV full-length E and N-terminal peptide Western blot. Lane 1: marker; lane 2: without β-mercaptoethanol; lane 3: Hepatitis C p7; lane 4: with β-mercaptoethanol. (A) About 150 μg/lane full-length E protein detected with anti-N terminal polyclonal antibody. (B) About 30 μg/lane E protein N-terminal peptide detected with anti-N terminal polyclonal antibody.

Fig. 5

SARS-CoV E protein ion channel activity is blocked by anti-N-terminal affinity-purified polyclonal antibody. CIS chamber contained 500 mM NaCl, 5 mM HEPES buffer (pH 7.2); the TRANS chamber contained 50 mM NaCl, 5 mM HEPES buffer (pH 7.2). Representative current traces at holding potential of −60 mV. The closed state is shown as a broken line, openings are deviations from the line. Scale bars are 0.15 s and 2 pA. (A) E protein ion channel activity before addition of antibody. (B) E protein ion channel activity after addition of ≤20 μg anti-N-terminal antibody to the CIS chamber (n = 4). (C) E protein ion channel activity before addition of antibody. (D) E protein ion channel activity after addition of ≤20 μg anti-N-terminal antibody to the TRANS chamber (n = 4).