Transcription of a Recombinant Bunyavirus RNA Template by Transiently Expressed Bunyavirus Proteins

• Published in: Virology (New York, N.Y.) N.Y.), 1995-08, Vol.211 (1), p.133-143
• Main Authors: Dunn, Ewan F., Pritlove, David C., Jin, Hong, Elliott, Richard M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; bunyavirus; Cell Line; Cercopithecus aethiops; Chloramphenicol O-Acetyltransferase - analysis; Chloramphenicol O-Acetyltransferase - biosynthesis; Kidney; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Open Reading Frames; Orthobunyavirus - genetics; Orthobunyavirus - metabolism; Plasmids; Point Mutation; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Restriction Mapping; RNA - biosynthesis; RNA - metabolism; RNA, Viral - biosynthesis; RNA, Viral - metabolism; Templates, Genetic; Transcription, Genetic; Transfection; Vaccinia virus; Viral Proteins - biosynthesis
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1006/viro.1995.1386

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 at 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 s detailed genetic analysis of the factors involved in bunyavirus transcription.