A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA.
We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells. Two recombinant baculoviruses carrying cDNA of bacteriophage T7 RNA polymerase, with or without a nuclear localization signal, under the control of a mammalian promoter were constructed. High level expression of T7 RNA polymerase was observed in various mammalian cell lines after infection with the recombinant baculoviruses. After transfection of plasmids containing the luciferase gene under the control of the T7 promoter, high luciferase activity was detected in cells infected with the recombinant baculoviruses. We also constructed a plasmid containing an entire cDNA clone of type 1 poliovirus under the T7 promoter. Two days after transfection of the plasmid into the cells infected with the recombinant baculoviruses, a high titer of poliovirus was recovered. The use of the recombinant baculoviruses did not cause any cytopathic effects even at a high multiplicity of infection. The lack of replication ability and low toxicity are the advantageous features of the hybrid baculovirus-T7 polymerase system in comparison with the widely used vaccinia-T7 polymerase system for gene expression and recovery of infectious viruses from its cDNA.
Yap
C C
CC
Department of Virology II, National Institute of Health, Tokyo, Japan.
Ishii
K
K
Aoki
Y
Y
Aizaki
H
H
Tani
H
H
Shimizu
H
H
Ueno
Y
Y
Miyamura
T
T
Matsuura
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D017955:Poliovirus / Q000235:genetics*; D014764:Viral Proteins; D014779:Virus Replication
D018076:DNA, Complementary; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases