Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins.
We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription.
Dunn
E F
EF
Institute of Virology, University of Glasgow, Scotland, United Kingdom.
Pritlove
D C
DC
Jin
H
H
Elliott
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D016851:Orthobunyavirus / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D012313:RNA / Q000096:biosynthesis / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus; D014764:Viral Proteins / Q000096:biosynthesis*
D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; C039919:RNA, recombinant; D011994:Recombinant Proteins; D014764:Viral Proteins; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase