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.

All coronaviruses encode a small hydrophobic envelope (E) protein, which mediates viral assembly and morphogenesis by an unknown mechanism. We have previously shown that the E protein from Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) forms cation-selective ion channels in planar lipid bilayers (Wilson, L., McKinlay, C., Gage, P., Ewart, G., 2004. SARS coronavirus E protein forms cation-selective ion channels. Virology 330(1), 322-331). We now report that three other E proteins also form cation-selective ion channels. These E proteins were from coronaviruses representative of taxonomic groups 1-3: human coronavirus 229E (HCoV-229E), mouse hepatitis virus (MHV), and infectious bronchitis virus (IBV), respectively. It appears, therefore, that coronavirus E proteins in general, belong to the virus ion channels family. Hexamethylene amiloride (HMA)--an inhibitor of the HIV-1 Vpu virus ion channel--inhibited the HCoV-229E and MHV E protein ion channel conductance in bilayers and also inhibited replication of the parent coronaviruses in cultured cells, as determined by plaque assay. Conversely, HMA had no antiviral effect on a recombinant MHV with the entire coding region of E protein deleted (MHVDeltaE). Taken together, the data provide evidence of a link between inhibition of E protein ion channel activity and the antiviral activity of HMA.

Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltageclamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carded by sodium ions because it was blocked by TIX, decreased in amplitude when extraceUular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at ~-40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 • 0.05 nA (mean • 1 SEM) and -0.21 • 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = g~,/{1 + exp[(V' -V)/k)]}. The average g~, was 7.8 • 1.1 nS in the 21 neurons in slices and 4.4 • t.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at >50% of its normal amplitude when the transient current was completely inactivated.

A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents £owing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca + to solutions made channels formed by p7 less potassium-or sodium-selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV-1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents. ß

1. A persistent inward current activated by depolarization was recorded using the whole‐cell, tight seal technique in rat isolated cardiac myocytes. The amplitude of the inward current increased when cells were exposed to a solution with low oxygen tension. 2. The persistent inward current had the characteristics of the persistent Na+ current described previously in rat ventricular myocytes: it was activated at negative potentials (‐70 mV), reversed close to the equilibrium potential for Na+ (ENa), was blocked by TTX and was resistant to inactivation. 3. Persistent single Na+ channel currents activated by long (200‐400 ms) depolarizations were recorded in cell‐attached patches on isolated ventricular myocytes. Hypoxia increased the frequency of opening of the persistent Na+ channels. 4. Persistent Na+ channels recorded during hypoxia had characteristics similar to those of persistent Na+ channels recorded at normal oxygen tensions. They had a null potential at ENa, their amplitude varied with [Na+], they were resistant to inactivation and their mean open time increased with increasing depolarization. 5. The persistent Na+ channels in cell‐attached patches were blocked by TTX (50 microM) in the patch pipette and by lidocaine (100 microM). 6. It was concluded that hypoxia increases the open probability of TTX‐sensitive, inactivation‐resistant Na+ channels. The voltage dependence of these channels, and their greatly increased activity during hypoxia, suggest that they may play an important role in the generation of arrhythmias during hypoxia.