Synthesis of the poliovirus polypeptide 3AB in bacterial cells results in an increase in membrane permeability. The alterations observed resemble those elicited by bacteriophage lytic proteins, which are presumed to cause pore formation in biological membranes. This property has been exploited in the development of an in vivo screening system that allows morphological differentiation of Escherichia coli clones expressing either wild-type 3AB or variant 3AB proteins lacking the ability to permeabilize bacteria. Expression of the wild-type 3AB gene in the presence of a chromogenic β-galactosidase substrate causes E. coli clones to stain dark blue. In contrast, bacterial mutants that synthesize 3AB proteins with alterations in the hydrophobic domain lack pore-forming activity and stain to a light blue colour, allowing differentiation from wild-type clones. This phenotypic property correlates with the rate of entry of the β-galactosidase substrate into the bacteria. The method developed here was used to screen more than 8000 E. coli clones after random PCR mutagenesis of the poliovirus 3AB gene. Our results show the existence of three different domains involved in the permeabilizing activity of 3AB protein. Twenty individual amino acid substitutions were identified in clones that showed the mutant phenotype and such bacteria displayed different reduced levels of permeability towards ONPG, hygromycin B, lysozyme and uridine. The procedure reported here may be of general interest to understand structure-function relationships in other eukaryotic proteins known to form pores.
CarrascoL.,
CastrilloJ. L.1987; The regulation of translation in picomavirus-infected cells. In Mechanisms of Viral Toxicity in Animal Cells pp 115–146 Edited by
CarrascoL.
Boca Raton: CRC Press;
CarrascoL.,
PerezL.,
IrurzunA.,
LamaJ.,
Martinez-AbarcaF.,
RodriguezP.,
GuineaR.,
CastrilloJ. L.,
SanzM. A.,
AyalaM. J.1993; Modification of membrane permeability by animal viruses. In Regulation of Gene Expression in Animal Viruses pp 283–305 Edited by
CarrascoL.,
SonenbergN.,
WimmerE.
New York: Plenum Press;
DubendorffJ. W.,
StudierF. W.1991; Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology 219:45–59
GiachettiC. S.,
HwangS.,
SemlerB. L.1992; cis-Acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication. Journal Virology 66:6045–6057
HarberJ.,
WimmerE.1993; Aspects of the molecular biology of poliovirus replication. In Regulation of Gene Expression in Animal Viruses pp 189–224 Edited by
CarrascoL.,
SonenbergN.,
WimmerE.
New York: Plenum Press;
LamaJ.,
CarrascoL.1992a; Inducible expression of a toxic poliovirus membrane protein in Escherichia coli. Comparative studies using different expression systems based on T7 promoters. Biochemical and Biophysical Research Communications 188:972–981
LamaJ.,
CarrascoL.1992b; Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A. Journal of Biological Chemistry 267:15932–15937
LamaJ.,
PaulA. V.,
HarrisK. S.,
WimmerE.1994; Properties of purified recombinant poliovirus protein 3AB as substrate for viral proteinases and as a cofactor for RNA polymerase 3Dpol. Journal of Biological Chemistry 269:66–70
LiebigH.,
SkernT.,
LudererM.,
SommergruberW.,
BlaasD.,
KuechlerD.1991; Proteinase trapping: screening for viral proteinase mutants by a complementation. Proceedings of the National Academy of Sciences USA 88:5979–5983
SullengerB. A.,
GallardoH. F.,
UngersG. E.,
GilboaE.1990; Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 63:601–608
TakedaN.,
KuhnR. J.,
YangC. F.,
TakegamiT.,
WimmerE.1986; Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. Journal of Virology 60:43–53
TakegamiT.,
KuhnR. J.,
AndersonC. W.,
WimmerE.1983a; Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus. Proceedings of the National Academy of Sciences USA 80:7447–7451