In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA.
A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection of HeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.
Yamanaka
K
K
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
Ogasawara
N
N
Yoshikawa
H
H
Ishihama
A
A
Nagata
K
K
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005818:Genetic Engineering; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D012367:RNA, Viral; D012261:Ribonucleoproteins; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases