A Bunyamwera Virus Minireplicon System in Mosquito Cells

Abstract

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 promotercontaining 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.

Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

“…Five hours later, the transfection mixture was removed and replaced with 2 ml of growth medium. At 24 h posttransfection, the supernatant was clarified by centrifugation for 3 min at 1,700 rpm, and 1.5 ml was used to infect BSR-T7/5 cells that had been transfected with pTM1-BUNN (0.1 g) and pTM1-BUNL (0.2 g) for 5 h. Renilla luciferase activity was measured after 24 h incubation using the dual-luciferase assay kit (Promega) as described previously (20). The level of Renilla luciferase activity of the infected cells was used as a measure of infectious VLP production.…”

Role of the Cytoplasmic Tail Domains of Bunyamwera Orthobunyavirus Glycoproteins Gn and Gc in Virus Assembly and Morphogenesis

“…At 24 h posttransfection, the supernatant was used to infect new monolayers of BSR-T7/5 cells. Renilla luciferase activity was measured after 24 h of incubation, using a dual-luciferase assay kit (Promega) as described previously (24).…”

Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication

“…Subgenomic RNAs containing virus -inducible reporter genes have been described for several RNA viruses (Kohl et al, 2004;Lo et al, 2003;Muhlberger et al, 1998;Olivo et al, 1998;Olivo et al, 1994) and a test kit utilizing a herpes simplex type 1-responsive gene is commercially available (Stabell et al, 1993;Tebas et al, 1998). The inherent enzymatic or fluorescence activity of the reporter gene precludes the need for antigenor sequence-specific reagents and the use of cis-acting promoter elements that are highly conserved among virus strains provides the requisite specificity.…”