A bunyamwera virus minireplicon system in mosquito cells.
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.
Kohl
Alain
A
Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, Scotland, United Kingdom.
Hart
Timothy J
TJ
Noonan
Carol
C
Royall
Elizabeth
E
Roberts
Lisa O
LO
Elliott
Richard M
RM
D000330:Aedes / Q000821:virology*; D000818:Animals; D002042:Bunyamwera virus / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012093:Replicon; D014158:Transcription, Genetic; D014764:Viral Proteins; D014779:Virus Replication
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases