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 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.

FIG. 1.

(A) Components of a T7 RNA polymerase-based BUN-minireplicon system in mosquito cells. The L, N, S, NSs, or firefly luciferase (FFLuc) ORF was cloned into pT7AcCat to produce the pT7Ac constructs used in this study. 5′Ac and 3′Ac, 5′ and 3′ translational enhancers of the baculovirus p10 gene; φ10, T7 RNA polymerase promoter. (B) Induction of firefly luciferase activity in mosquito cells expressing T7 RNA polymerase (C6-IBT7/3) or in parental cells (C6/36). Cells were either transfected with 100 ng of pT7AcLuc (bar 1) or of pT7AcCat (bar 2) or were not transfected (bar 3). (C) Analysis of various IRESs and translational enhancer elements in C6-IBT7/3 mosquito cells expressing T7 RNA polymerase. Monocistronic reporter constructs containing either the RhPV IRES in the sense (pSP72Δ1luc) or antisense (pSP72 Δ1asluc) orientation, the baculovirus p10 gene 5′ and 3′ translational enhancers (pT7AcLuc), or the EMCV IRES (pTM1-FF-Luc) were used. C6-IBT7/3 cells were transfected with 1 μg of the indicated plasmid. Firefly luciferase activities (expressed here as fold induction) were determined at 24 h posttransfection and normalized against an internal standard (the φ10 promoter-containing construct expressing Renilla luciferase). (D) Dual-reporter constructs containing either the RhPV IRES (pGEMCATΔ1luc), the EMCV IRES (pGEMCATEMCV-Luc), or no IRES element (PGEMCATluc), as indicated. C6-IBT7/3 cells were transfected with 1 μg of the indicated plasmid. Firefly luciferase activities (expressed here as fold induction) were determined at 24 h posttransfection and normalized against an internal standard (the φ10 promoter-containing construct expressing Renilla luciferase).

FIG. 2.

Minireplicon activities at 28, 33, or 37°C in mosquito cells expressing T7 RNA polymerase. C6-IBT7/3 cells were transfected with minireplicon components pT7AcL, pT7AcN, pT7riboBUNMREN(−), and pT7AcLuc (internal transfection control). Renilla luciferase (Ren luc) activities were determined at 24 h posttransfection and normalized against the internal transfection control. Activities are expressed as fold induction.

FIG. 3.

Analysis of promoter strength in mammalian BSR-T7/5 or mosquito C6-IBT7/3 cells. Cells were transfected with pTM1-based (for mammalian cells) or pT7Ac-based (for mosquito cells) L and N expression vectors and the appropriate minigenome plasmid vector pT7riboBUNMREN(−), pT7riboBUNLREN(−), pT7riboBUNSREN(−), or pT7riboBUNSREN(−)mut16. pT7AcLuc was cotransfected as an internal control to standardize luciferase activities. (−) control, negative control, showing background minigenome activity in the absence of functional L protein. Activities are expressed in light units or as fold induction of Renilla luciferase (Ren luc) activity.

FIG. 4.

Effects of NSs on minireplicon activity in mosquito and mammalian cells. Cells were transfected with pTM1-based or pT7Ac-based L expression vectors as appropriate and the minigenome plasmid pT7riboBUNMREN(−), along with plasmids expressing either N and NSs (pTM1-BUNS or pT7AcS) or N alone (pTM1-BUNN or pT7AcN), as indicated. pT7AcLuc was cotransfected as an internal control to standardize luciferase activities. (−) control, negative control without functional L protein. The amounts of DNA transfected were kept constant by adding the corresponding empty plasmid if necessary. Activities are expressed in light units (for BSR-T7/5) or as fold induction of Renilla luciferase (Ren luc) activity (for C6-IBT7/3). (A) Control experiment in BSR-T7/5 cells transfected with N- or N-plus-NSs-expressing pTM1- or pT7Ac-based expression plasmids and minireplicon components as described above. (B) C6-IBT7/3 cells transfected with N- or N-plus-NSs-expressing pT7Ac-based expression plasmids and minireplicon components as described above. (C) Control experiment in BSR-T7/5 cells transfected with NSs-expressing pTM1- or pT7Ac-based plasmids and minireplicon components as described above. (D) C6-IBT7/3 cells transfected or not with the pT7AcNSs plasmid and minireplicon components as described above. (E) Expression of NSs protein in transfected mosquito cells. C6-IBT7/3 cells were either transfected with 1 (lane 1) or 2 (lane 2) μg of pT7AcNSs or left untransfected (lane 3). Cells infected with wtBUN (lane 4) were used as a positive control. Following SDS-PAGE and blotting to a membrane, NSs protein was detected by using an NSs-specific antibody; different exposures were needed to visualize NSs in transfected and infected cells. The positions of NSs and molecular size standards (in kilodaltons) are indicated.

FIG. 5.

Effects of NSs expression on protein synthesis. (A) Metabolic labeling of BUN-infected mammalian BHK-21 and mosquito C6/36 cells. Cells were infected at a multiplicity of infection of 5 by wtBUN or BUNdelNSs 9a; at 18 h postinfection, they were labeled with 50 μCi of [³⁵S]methionine for 2 h. Cell extracts were analyzed by SDS-PAGE (18% acrylamide gel). (B) Effects of NSs on transient reporter gene expression in mammalian BSR-T7/5 and mosquito C6-IBT7/3 cells. Cells were cotransfected with phRL-CMV (Promega) (0.5 μg per well) and pT7AcNSs at various concentrations (5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 μg per well). Renilla luciferase reporter gene activities were determined at 16 h posttransfection by using the single Renilla luciferase assay kit (Promega).