Semliki Forest virus 6K protein modifies membrane permeability after inducible expression in Escherichia coli cells.
Semliki Forest virus encodes a small protein, known as 6K, that is associated with cellular membranes in the infected cells. This protein has been cloned and expressed in an inducible manner using pET vectors in Escherichia coli cells. Two different plasmids have been utilized; either the 6K gene is placed directly under the T7 promoter (pET3-6K) or the lac operator is located between the T7 promoter and the 6K gene (pET11-6K). In both systems, efficient synthesis of the 6K protein is achieved by induction with isopropyl-1-thio-beta-D-galactopyranoside plus rifampicin. The synthesis of the 6K protein is very toxic for E. coli causing increased membrane permeability and cell lysis as shown by alterations in permeability to either choline or hygromycin B. These results indicate that the togavirus 6K is a membrane-active protein that shows structural and functional similarities to poliovirus 3A protein. The function that the 6K protein could play during the virus replication cycle is discussed in the light of these findings.
Sanz
M A
MA
Centro de Biología Molecular, Universidad Autónoma, Madrid, Spain.
Pérez
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Carrasco
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D001483:Base Sequence; D002463:Cell Membrane Permeability; D002794:Choline / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D005814:Genes, Viral; D006921:Hygromycin B / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D012293:Rifampin / Q000494:pharmacology; D012672:Semliki forest virus / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D014759:Viral Envelope Proteins; D007544:Isopropyl Thiogalactoside; D006921:Hygromycin B; D002794:Choline; D012293:Rifampin