The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.
Coronaviruses are viral pathogens that cause a variety of lethal diseases in birds and mammals, and common colds in humans. In 2003, however, an animal coronavirus was able to infect humans and produced severe acute respiratory syndrome (SARS), causing a near pandemic. Such events are likely to reoccur in the future, and new antiviral strategies are necessary. A small coronavirus protein called ‘envelope’ is important for pathogenesis, affecting the formation of the viral envelope and the distribution of the virus in the body. In vitro studies have shown that synthetic coronavirus envelope proteins have channel activity that in some cases has been inhibited by the drug hexamethylene amiloride, but not by amiloride. In the present paper, we have characterized the structure responsible for this channel activity. We have also determined the binding site of the drug hexamethylene amiloride in the channel, and shown that amiloride has only a mild effect on the NMR signals from the protein. The validity of these results is supported using mammalian cells expressing full length SARS-CoV E, where channel activity was inhibited by hexamethylene amiloride, but only mildly by amiloride. The structural model described for this channel provides a valuable insight into coronavirus envelope protein ion channel activity, and could serve as a platform for the development of novel anti-viral drugs.