Development of a novel Borna disease virus reverse genetics system using RNA polymerase II promoter and SV40 nuclear import signal.
Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that replicates and transcribes in the nucleus of infected cells. Therefore, efficient synthesis of BDV RNA in the nucleus is critical for the development of a reverse genetics system for this virus. Here, we report the development of such a system using the RNA polymerase II (Pol II) promoter. The BDV minigenome cDNA was flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences and inserted downstream of the Pol II promoter. To improve the efficacy of minigenome expression, we estimated the effects of several signal sequences within the minigenome constructs. We found that insertion of the SV40 nuclear import sequence into the Pol II constructs significantly enhances the replication of the minigenome even in cells lacking the SV40 large T antigen. This novel system is theoretically applicable to any mammalian cell line and would be valuable for analyzing host- or cell-type-dependent differences in BDV replication and production. We could demonstrate here the cell-type-dependent inhibitory effect of the viral protein X on BDV polymerase activity. This system may be useful for various research fields not only of BDV but also of other negative-sense RNA viruses.
Yanai
Hideyuki
H
Department of Virology, Research Institute for Microbial Diseases (BIKEN), Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Hayashi
Yohei
Y
Watanabe
Yohei
Y
Ohtaki
Naohiro
N
Kobayashi
Takeshi
T
Nozaki
Yasutoshi
Y
Ikuta
Kazuyoshi
K
Tomonaga
Keizo
K
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001891:Borna disease virus / Q000235:genetics*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009842:Oligopeptides / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics; D014162:Transfection; D014709:Vero Cells
D009842:Oligopeptides; D016337:RNA, Catalytic; C402304:SV40 nuclear localization peptide; D012319:RNA Polymerase II