Experimental Study on 3a from SARS-CoV within Artificial Lipid Bilayers

• Main Authors: Tze-Hsiang Chien, 簡子翔
• Other Authors: Wolfgang B. Fischer
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/89403186587096742222

碩士 === 國立陽明大學 === 生醫光電工程研究所 === 99 === The outbreak of a disease in China in 2002 which showed a severe affection on the respiratory system was later termed Severe Acute Respiratory Syndrome (SARS-CoV). The cause of the disease was identified as a novel coronavirus(1) which causes the common cold in humans and a variety of lethal diseases in birds and mammals. The outbreak caused alert to the whole of the world. SARS-CoV has a large single-channel positive(2) strand RNA genome that contains 14 open reading frames(3) (ORFs) comprising a spike protein, small envelope protein, membrane protein and nucleocapsid protein(4). SARS 3a protein is one of the ORFs in the viral genome has no homologue in other known corona viruses. Some researching say that this protein of 3A has low similarly with other known coronavirus protein(2). Studies have shown that the protein encoded by ORF 3a is located at the cell surface(5) of infected cells exhibiting channel activity. It is suggested that its expression plays an important role in the life cycle and that it will form a cation channel(6). The ion channel of 3a protein is permeable for monovalent cations with higher permeability for K+ ions than to Na+ ions(2). The 3a protein is composed of three transmembrane domains(7) (TMDs) and forms covalently linked dimers which assemble into tetramers(8). In this study it is investigated whether the individual TMDs of 3a if placed individually into a lipid membrane would self-assemble and form a functional channel. The studies are compared with data from membrane fractions of the whole length protein expressed in italics Escherichia Coli, purified and reconstituted into artificial lipid bilayers. The investigations show that the full length protein forms cation-selective ion channels. The data shed light on the putative roles of the individual TMDs. With TMD2 and TMD3 exhibiting activity the role of TMD1 is most likely not pore lining. Channel activity is almost restored when mixing the peptides in a 1:1:1 ratio. The consequences for modeling the channel are discussed. The bilayer recordings show potassium channel activity with a main conductance level of around 10 pS. Our results also suggest that 3A protein conducts some calcium and chloride. In recent paper, there has been a report research for the development of novel therapeutic agents. By inhibiting the SARS 3A channel with the already known drug, Emodin(9), our experiments can provide more complete insights into the dynamics of SARS 3A protein.