Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA.
Plasmids have been constructed that contain DNA sequences that direct the expression of the poliovirus RNA-dependent RNA polymerase, in the form of recombinant fusion proteins. Inclusion of an additional gene for the poliovirus protease results in cleavage of the fusion protein to yield a 52-kDa, enzymatically active, polymerase protein, apparently identical to the functional enzyme isolated from virus-infected HeLa cells. A large amount of polymerase protein accumulates as particulate or insoluble material in bacteria, and this protein has little or no activity. However, significant amounts of soluble, active enzyme are recovered, such that the resulting specific activity of crude bacterial extracts is greater than that obtained from virus-infected HeLa cells. Purification of the enzyme from Escherichia coli is readily accomplished, and yields a preparation that will copy poliovirion RNA as template, in the presence of oligo(U) primer. The availability of cloned DNA sequences encoding catalytically active RNA polymerase will allow genetic manipulations to initiate structure-function studies of this enzyme.
Rothstein
M A
MA
Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City 84132.
Richards
O C
OC
Amin
C
C
Ehrenfeld
E
E
D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014779:Virus Replication
D012367:RNA, Viral; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases