ABSTRACT
Bunyaviruses are trisegmented, negative-sense RNA viruses. Previously, we described a rescue system to recover infectious Bunyamwera virus (genus Orthobunyavirus) entirely from cloned cDNA (Bridgen, A. and Elliott, R.M. (1996) Proc. Nat. Acad. Sci. USA 93, 15400-15404) utilizing a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase to drive intracellular transcription of transfected T7 promoter-containing plasmids. Here we report efforts to improve the efficiency of the system by comparing different methods of providing T7 polymerase. We found that a BHK-derived cell line BSR-T7/5 that constitutively expresses T7 RNA polymerase supported efficient and reproducible recovery of Bunyamwera virus, routinely generating >10(7) pfu per rescue experiment. Furthermore, we show that the virus can be recovered from transfecting cells with just three plasmids that express full-length antigenome viral RNAs, greatly simplifying the procedure. We suggest that this procedure should be applicable to viruses in other genera of the family Bunyaviridae and perhaps also to arenaviruses.
Subject(s)
DNA, Complementary , Orthobunyavirus/genetics , Orthobunyavirus/physiology , Virology/methods , Virus Replication , Animals , Cell Line , Cricetinae , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Plasmids , RNA, Viral/genetics , RNA, Viral/metabolism , Viral ProteinsABSTRACT
Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.