Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A.
The poliovirus nonstructural proteins 2B, 2C, 2C3A, 2C3AB, 3A, and 3AB have been cloned and efficiently expressed in Escherichia coli cells. Each individual protein, or combinations of some of them, were cloned using polymerase chain reaction techniques and correspond to the genuine poliovirus protein plus an additional methionine. The system used to express them uses pET vectors containing the promoter of gene 10 of phage T7. Expression of protein 2C in BL21 (DE3) pLysS cells, which express the T7 lysozyme, is not toxic, and the bacteria synthesize this protein for several hours after induction. In contrast, the expression of proteins 2B, 3A, or 3AB is not tolerated by BL21 (DE3) pLysS cells which could make them only for a limited period of time. Protein 3AB was particularly toxic and induced a rapid lysis of the recombinant clone after its induction with isopropyl-1-thio-beta-D-galactopyranoside alone or with both isopropyl-1-thio-beta-D-galactopyranoside and rifampicin. Further analyses showed that 3AB induced profound modifications in membrane permeability to o-nitrophenyl-beta-D-galactopyranoside, labeled uridine, and nonpermeant translation inhibitors. Cloning and expression of proteins 2B, 3A, and 3AB in BL21 (DE3) cells that do not contain the T7 lysozyme lead to a more sustained expression of these proteins without detectable cell lysis. Changes in permeability to low molecular weight compounds such as radioactive uridine, o-nitrophenyl-beta-D-galactopyranoside, and hygromycin B readily appeared upon induction of 2B, 3A, and 3AB. Our results indicate that the poliovirus nonstructural polypeptides 2B and 3A (or 3AB) are lytic for the bacteria. In fact, both proteins 2B and 3A contain hydrophobic domains in a potential amphipathic helix; this is one characteristic shared with a number of membrane-active peptides.
Lama
J
J
Centro de Biología Molecular, Universidad Autónoma de Madrid, Canto Blanco, Spain.
Carrasco
L
L
D001345:Autoradiography; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012293:Rifampin / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013604:T-Phages / Q000235:genetics; D014529:Uridine / Q000378:metabolism; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D017361:Viral Nonstructural Proteins
D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D008715:Methionine; D012293:Rifampin; D014529:Uridine