title: Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs.
abstract: We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5'- and 3'-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.
authors.affiliation: Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, S-17177 Stockholm, Sweden.
authors.forename: R
authors.initials: R
authors.lastname: Flick
authors.forename: R F
authors.initials: RF
authors.lastname: Pettersson
mesh_terms: D000818:Animals; D001483:Base Sequence; D002044:Bunyaviridae Infections / Q000821:virology*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012318:RNA Polymerase I / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D018066:Uukuniemi virus / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012318:RNA Polymerase I