Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

Pervushin, Konstantin; Tan, Edward; Parthasarathy, Krupakar; Lin, Xin; Jiang, Feng Li; Yu, Dejie; Vararattanavech, Ardcharaporn; Soong, Tuck Wah; Liu, Ding Xiang; Torres, Jaume

The full-length SARS-CoV E protein gene was cloned into pIRES-AcGFP1 (Clonetech) vector by using the restriction enzymes BglII and PstI. The identity of the insert was confirmed by DNA sequencing. In a 35 mm Petri dish, 1.65 mg of human SARS-CoV E cDNA was transiently transfected into HEK-293 cells using the standard calcium phosphate method [70] . The vector pIRES-AcGFP1 was also transiently transfected in separate experiments as a control. Another control was the use of untransfected HEK-293 cells. The cells were grown for 36-48 h in a 5% CO 2 incubator at 37uC before whole-cell patch clamp recordings. 21 ). The voltages were uncorrected for a 29 mV junction potential, and actual voltage is obtained by subtracting 9 mV from the reported values. Whole-cell currents, obtained under voltage clamp with an Axopatch Multiclamp700B amplifier (Axon Instruments), were filtered at 1-5 kHz and sampled at 5-50 kHz. The access resistance R a (usually less than 20 MV) and the capacitive transients were not compensated. Stock solutions of amiloride and HMA (Sigma) at 100 mM were prepared in 50% DMSO:50% methanol. To determine if the amiloride derivatives blocked SARS-CoV E protein ion channel conductance in HEK-293 cells, after ion channel currents were detected, 10 mM of the drug diluted in bath solution was applied to the cell.